Polyvalent protein complex

ABSTRACT

The invention provides for a polyvalent protein complex (PPC) comprising two polypeptide chains generally arranged laterally to one another. Each polypeptide chain typically comprises 3 or 4 “v-regions”, which comprise amino acid sequences capable of forming an antigen binding site when matched with a corresponding v-region on the opposite polypeptide chain. Up to about 6 “v-regions” can be used on each polypeptide chain. The v-regions of each polypeptide chain are connected linearly to one another and may be connected by interspersed linking regions. When arranged in the form of the PPC, the v-regions on each polypeptide chain form individual antigen binding sites.

This application claims priority to U.S. Provisional Application No.60/464,532, filed Apr. 22, 2003, and 60/525,391, filed Nov. 24, 2003,the contents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to polyvalent protein complexes, includingtrivalent bispecific proteins, useful for the treatment and diagnosis ofdiseases, and to methods of producing such proteins.

BACKGROUND OF THE INVENTION

Throughout this specification, various patents, published applicationsand scientific references are cited to describe the state and content ofthe art. Those disclosures, in their entireties, are hereby incorporatedinto the present specification by reference.

The present invention is directed to a novel protein structures, termeda “polyvalent protein complex” or PPC, that comprise three or fourantigen binding sites (ABS). These PPC comprise novel properties, suchas trivalence and tetravalence, when compared to immunoglobulins and cansubstitute for immunoglobulins or other engineered antibodies inapplications such as diagnosis, detection, and therapy of normal(ectopic) or diseased tissues. These diseased tissues include cancers,infections, autoimmune diseases, cardiovascular diseases, andneurological diseases. Normal tissues can be detected and/or ablated,such as when they are ectopic (misplaced, such as parathyroid, thymus,endometrium) or if they need to be ablated as a therapy measure (e.g.,bone marrow ablation in cancer therapies).

Discrete V_(H) and V_(L) domains of antibodies produced by recombinantDNA technology may pair with each other to form a heterodimer(recombinant Fv fragment) with binding capability (U.S. Pat. No.4,642,334). However, such non-covalently associated molecules are notsufficiently stable under physiological conditions to have any practicaluse. Cognate V_(H) and V_(L) domains can be joined with a peptide linkerof appropriate composition and length (usually consisting of more than12 amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405. Reduction of the peptide linkerlength to less than 12 amino acid residues prevents pairing of V_(H) andV_(L) domains on the same chain and forces pairing of V_(H) and V_(L)domains with complementary domains on other chains, resulting in theformation of functional multimers. Polypeptide chains of V_(H) and V_(L)domains joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are in favor, but the exact patterns of oligomerizationappear to depend on the composition as well as the orientation ofV-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to thelinker length. Monospecific diabodies, triabodies, and tetrabodies withmultiple valencies have been obtained using peptide linkers consistingof 5 amino acid residues or less. Bispecific diabodies, which areheterodimers of two different polypeptides, each polypeptide consistingof the V_(H) domain from one antibody connected by a short peptidelinker to the V_(L) domain of another antibody, have also been madeusing a dicistronic expression vector that contains in one cistron arecombinant gene construct comprising V_(H1)-linker-V_(L2) and in theother cistron a second recombinant gene construct comprisingV_(H2)-linker-V_(L1). (Holliger et al., Proc. Natl. Acad. Sci. USA(1993) 90: 6444-6448; Atwell et al., Molecular Immunology (1996) 33:1301-1302; Holligeret al., Nature Biotechnology (1997) 15: 632-631;Helfrich et al., Int. J. Cancer (1998) 76: 232-239; Kipriyanov et al.,Int. J. Cancer (1998) 77: 763-772; Holiger et al., Cancer Research(1999) 59: 2909-2916]. More recently, a tetravalent tandem diabody(termed tandab) with dual specificity has also been reported (Cochloviuset al., Cancer Research (2000) 60: 4336-4341]. The bispecific tandab isa dimer of two homologous polypeptides, each containing four variabledomains of two different antibodies (V_(H1), V_(L1), V_(H2), V_(L2))linked in an orientation to facilitate the formation of two potentialbinding sites for each of the two different specificities uponself-association.

Methods of manufacturing monospecific diabodies, monospecifictriabodies, monospecific tetrabodies and bispecific diabodies by varyingthe length of the peptide linker as described above are disclosed inU.S. Pat. No. 5,844,094, U.S. Pat. No. 5,837,242, and WO 98/44001.

Alternative methods of manufacturing multispecific and multivalentantigen-binding proteins from V_(H) and V_(L) domains are disclosed inU.S. Pat. No. 5,989,830 and U.S. Pat. No. 6,239,259. Such multivalentand multispecific antigen-binding proteins are obtained by expressing adicistronic vector which encodes two polypeptide chains, with onepolypeptide chain consisting of two or more V_(H) domains (from the sameor different antibodies) connected in series by a peptide linker and theother polypeptide chain consisting of complementary V_(L) domainsconnected in series by a peptide linker.

Increasing the valency of a binding protein is of interest as itenhances the functional affinity of that protein due to the avidityeffect. The increased affinity enables the resulting protein to bindmore strongly to target cells. Furthermore, the multivalency may, viacrosslinking, induce growth inhibition of target cells (Ghetie, et al,Blood, 97: 1392-8, 2001) or facilitate internalization (Yarden, Proc.Natl. Acad. Sci., USA, 94: 9637, 1990), either property is desirable foran anti-tumor agent. The present invention addresses the continuous needto develop multivalent, multispecific agents for use in therapeutic anddiagnostic applications.

Another area of the present invention is in the field of bio-assays.Virtually every area of biomedical sciences is in need of a system toassay chemical and biochemical reactions and determine the presence andquantity of particular analytes. This need ranges from the basic scienceresearch lab, where biochemical pathways are being mapped out and theirfunctions correlated to disease processes, to clinical diagnostics,where patients are routinely monitored for levels of clinically relevantanalytes. Other areas include pharmaceutical research, militaryapplications, veterinary, food, and environmental applications. In allof these cases, the presence and quantity of a specific analyte or groupof analytes, needs to be determined.

For analysis in the fields of chemistry, biochemistry, biotechnology,molecular biology and numerous others, it is often useful to detect thepresence of one or more molecular structures and measure binding betweenstructures. The molecular structures of interest typically include, butare not limited to, cells, antibodies, antigens, metabolites, proteins,drugs, small molecules, proteins, enzymes, nucleic acids, and otherligands and analytes. In medicine, for example, it is very useful todetermine the existence of a cellular constituents such as receptors orcytokines, or antibodies and antigens which serve as markers for variousdisease processes, which exists naturally in physiological fluids orwhich has been introduced into the system. Additionally, DNA and RNAanalysis is very useful in diagnostics, genetic testing and research,agriculture, and pharmaceutical development. Because of the rapidlyadvancing state of molecular cell biology and understanding of normaland diseased systems, there exists an increasing need for methods ofdetection, which do not require labels such as fluorophores orradioisotopes, are quantitative and qualitative, specific to themolecule of interest, highly sensitive and relatively simple toimplement.

Numerous methodologies have been developed over the years to meet thedemands of these fields, such as Enzyme-Linked Immunosorbent Assays(ELISA), Radio-Immunoassays (RIA), numerous fluorescence assays, massspectroscopy, colorimetric assays, gel electrophoresis, as well as ahost of more specialized assays. Most of these assay techniques requirespecialized preparations, especially attaching a label or greatlypurifying and amplifying the sample to be tested. To detect a bindingevent between a ligand and an antiligand, a detectable signal isrequired which relates to the existence or extension of binding. Usuallythe signal is provided by a label that is conjugated to either theligand or antiligand of interest. Physical or chemical effects whichproduce detectable signals, and for which suitable labels exist, includeradioactivity, fluorescence, chemiluminescence, phosphorescence andenzymatic activity to name a few. The label can then be detected byspectrophotometric, radiometric, or optical tracking methods.

SUMMARY OF THE INVENTION

This invention provides a polyvalent protein complex (PPC), a dimer,comprising at least three antigen binding sites (ABS) in a linear array.The invention also provides a fusion PPC, which is a PPC chemicallybonded to a second molecule such as a conjugate. It is understood that“fusion PPC” is a subset of all PPC and that references to PPC in thisdisclosure is also meant to refer to “fusion PPC.”

The invention also provides a nucleic acid that encodes at least onepolypeptide of a PPC. A host cell that comprise the polypeptide is alsoan embodiment of the invention.

In addition, the invention also provides a method for reducing a symptomof a disorder, such as a cancer, an infection, a cardiological disorderor an autoimmune disorder by administering a PPC or fusion PPC to apatient.

Specifically, there is provided a polyvalent protein complex (PPC)containing a first and a second polypeptide chain, where the firstpolypeptide chain contains a polypeptide sequence represented, by theformula a₁-1₁-a₂-1₂-a₃, where a₁, a₂, and a₃ are immunoglobulin variabledomains and 1₁ and 1₂ are peptide linkers, and a₁ is N-terminal of a₂,which in turn is N-terminal of a₃, where the second polypeptide chaincontains a polypeptide sequence represented by the formulab₁-1₃-b₂-1₄-b₃, where b₁, b₂, and b₃ are immunoglobulin variable domainsand 1₃ and 1₄ are peptide linkers, and b₃ is N-terminal of b₂, which inturn is N-terminal of b₁, where the first and second polypeptide chaintogether form a complex containing at least three antigen binding sites,where each of the antigen binding sites contains a variable domain fromthe first polypeptide chain and a variable domain from the secondpolypeptide chain, and where each binding site contains animmunoglobulin heavy chain variable domain and an immunoglobulin lightchain variable domain.

Each polypeptide chain may further contain 1-3 additional immunoglobulinvariable domains, where each domain is linked via a peptide linker,where the first and second polypeptide chain together form a complexcontaining 4-6 antigen binding sites, and where each of the antigenbinding sites contains a variable domain from the first polypeptidechain and a variable domain from the second polypeptide chain. At leastone of the polypeptide chains may further contain an amino acid sequenceselected from the group consisting of a toxin, a cytokine, a lymphokine,a enzyme, a growth factor, and an affinity purification tag.

The complex may contain any of the possible combinations of bindingaffinities, for example, at least two of the antigen binding sites mayhave the same binding specificity, each of the antigen binding sites mayhave a different or the same binding specificity, the antigen bindingsites may have at least two different binding specificities, at least 3of the antigen binding sites may have different binding specificities,at least 4 of the antigen binding sites may have different bindingspecificities, the complex may contain at least 5 antigen binding siteswhere at least 5 of the binding sites have different bindingspecificities, or the complex may contain 6 antigen binding sites eachhaving a different binding specificity. In another example two of theantigen binding sites are specific for epitopes of tumor associatedantigens, and the third antigen binding sites is reactive with atargetable construct. In another example, two antigen binding sites arespecific for epitopes of tumor associated antigens, and the thirdantigen binding sites is reactive with a targetable construct, where theepitope on the targetable construct is a hapten. In still anothercomplex, the complex is bound to a first hapten on the construct and theconstruct further contains a second hapten capable of bindingsimultaneously to a second polyvalent protein complex.

In each of these examples, the complex may bind tumor associatedantigen, or antigens are selected from the group consisting of antigensassociated with carcinomas, melanomas, sarcomas, gliomas, leukemias andlymphomas, such as α-fetoprotein, A3, CA125, carcinoembryonic antigen(CEA), CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45, CD74, CD80,colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, folate receptor,HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia, IL-2, IL-6,insulin-like growth factor, KS-1, Le(y), MAGE, MUC1, MUC2, MUC3, MUC4,NCA66, necrosis antigens, PAM-4, placental growth factor, prostatic acidphosphatase PSA, PSMA, S100, T101, TAC, TAG-72, tenascin and/or VEGF.

In another example, the complex contains at least two tumor antigenbinding sites, where both tumor antigen binding sites are specific forCEA and where the third binding site is specific for the hapten,histamine-succinyl-glycine (HSG).

The polyvalent protein may be BS14HP, or hBS14, which may be bound toIMP 241, or IMP 245

In another embodiment, any of the complexes described above may be usedin a pretargeting method of treating or diagnosing or treating anddiagnosing a neoplastic condition by (a) administering to the subject acomplex as above, where two antigen binding sites are directed to atumor associated antigen, and one antigen binding sites is directed to atargetable construct containing a bivalent hapten; (b) optionally,administering to the subject a clearing composition, and allowing thecomposition to clear the polyvalent complex from circulation; and (c)administering to the subject the targetable construct containing abivalent hapten, where the targetable construct further contains one ormore chelated or chemically bound therapeutic or diagnostic agents.

The diagnostic agent may be a radionuclide selected from the groupconsisting of ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd,¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and ⁹⁰Y or a combination thereof, which may bedetected, for example, by computed tomography (CT), single photonemission computed tomography (SPECT), or positron emission tomography(PET). The application may be for intraoperative diagnosis to identifyoccult neoplastic tumors. The targetable construct may contain one ormore image enhancing agents for use in magnetic resonance imaging (MRI),such as a metal selected from the group consisting of chromium (III),manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper(II), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III) anderbium (III). The targetable construct may contains one or more imageenhancing agents for use in ultrasound imaging.

The targetable construct may be a liposome with a bivalent HSG-peptidecovalently attached to the outside surface of the liposome lipidmembrane. The liposome may be gas filled.

The targetable construct may contain one or more radioactive isotopesuseful for killing neoplastic cells, such as ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag, ₁₁₁In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Acor a combination thereof.

The pretargeted therapy may be administered prior to, with or after oneor more therapeutic agents. The therapeutic agent may be a cytokine or achemotherapeutic agent, or a colony-stimulating growth factor. Thetherapeutic agent may be a chemotherapeutic agent selected from thegroup consisting of taxanes, nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes; folic acidanalogs, pyrimidine analogs, purine analogs, vinca alkaloids,antibiotics, enzymes, platinum coordination complexes, substituted urea,methyl hydrazine derivatives, adrenocortical suppressants, andantagonists, or may be selected from the group consisting of steroids,progestins, estrogens, antiestrogens, and androgens. The therapeuticagent may be a chemotherapeutic agent selected from the group consistingof azaribine, bleomycin, bryostatin-1, busulfan, carmustine,chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, dexamethasone,diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide,fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesteronecaproate, hydroxyurea, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,semustine streptozocin, tamoxifen, taxanes, taxol, testosteronepropionate, thalidomide, thioguanine, thiotepa, uracil mustard,vinblastine, and vincristine. The therapeutic agent may be a cytokineselected from the group consisting of interleukin-1 (IL-1), IL-2, IL-3,IL-6, IL-10, IL-12, interferon-alpha, interferon-beta, andinterferon-gamma, or may be a colony-stimulating growth factor selectedfrom the group consisting of granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF),erthropoietin and thrombopoietin.

Also provided is a method of treating a neoplastic disorder in asubject, by administering to the subject a “naked” polyvalent proteincomplex as described above, where at least one of the antigen bindingsites binds to an antigen selected from the group consisting of alphafetoprotein, A3, CA125, carcinoembryonic antigen (CEA), CD19, CD20,CD21, CD22, CD23, CD30, CD33, CD45, CD74, CD80, colon-specific antigen-p(CSAp), EGFR, EGP-1, EGP-2, folate receptor, HER2/neu, HLA-DR, humanchorionic gonadrotropin, Ia, IL-2, IL-6, insulin-like growth factor,KS-1, Le(y), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, necrosis antigens,PAM-4, placental growth factor, prostatic acid phosphatase PSA, PSMA,S100, T101, TAC, TAG-72, tenascin and VEGF.

The neoplastic disorder may be selected from the group consisting ofcarcinomas, sarcomas, gliomas, lymphomas, leukemias, and melanomas.

Also provided is a method for treating a B-cell malignancy, or B-cellimmune or autoimmune disorder in a subject, containing administering tothe subject one or more dosages of a therapeutic composition containinga polyvalent protein complex as described above and a pharmaceuticallyacceptable carrier.

Also provided is a method for treating a B-cell malignancy, or B-cellimmune or autoimmune disorder in a subject, by administering to thesubject one or more dosages of a therapeutic composition containing apolyvalent protein complex and a pharmaceutically acceptable carrier,where each antigen binding site binds a distinct epitope of CD19, CD20or CD22. The complex may be parenterally administered in a dosage of 20to 1500 milligrams protein per dose, or 20 to 500 milligrams protein perdose, or 20 to 100 milligrams protein per dose. The subject may receiverepeated parenteral dosages of 20 to 100 milligrams protein per dose, orrepeated parenteral dosages of 20 to 1500 milligrams protein per dose.In these methods, a sub-fraction of the polyvalent protein complex islabeled with a radioactive isotope, such as ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag, ₁₁₁In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac or acombination thereof.

Also provided is a method for detecting or diagnosing a B-cellmalignancy, or B-cell immune or autoimmune disorder in a subject, byadministering to the subject a diagnostic composition containing apolyvalent protein complex as above and a pharmaceutically acceptablecarrier, where each antigen binding site binds a distinct epitope ofCD19, CD20 or CD22, and where the complex is radiolabeled with aradionuclide selected from the group consisting of ¹⁸F, ⁵²Fe, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and ⁹⁰Y or a combinationthereof. Detection may be as described above. The application may be forintraoperative diagnosis to identify occult neoplastic tumors.

Also provided is a method for detecting or diagnosing a B-cellmalignancy, or B-cell immune or autoimmune disorder in a subject,containing administering to the subject a diagnostic compositioncontaining a polyvalent protein complex as above and a pharmaceuticallyacceptable carrier, where each antigen binding site binds a distinctepitope of CD19, CD20 or CD22, and where the complex is labeled with oneor more image enhancing agents for use in magnetic resonance imaging(MRI). The image enhancing agent may be as described above

Also provided is a method of diagnosing a non-neoplastic disease ordisorder, by administering to a subject suffering from the disease ordisorder a complex as above, where a detectable label is attached to thecomplex, and where one or more of the antigen binding sites is specificfor a marker substance of the disease or disorder. The disease ordisorder may be caused by a fungus, such as Microsporum, Trichophyton,Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans,Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis,and Candida albican, or a virus, such as human immunodeficiency virus(HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia-viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus, wart virus and blue tongue virus. The disease ordisorder may be caused by a bacterium, such as Anthrax bacillus,Streptococcus agalactiae, Legionella pneumophilia, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, and Mycobacterium tuberculosis, or a Mycoplasma. Thedisease or disorder may be caused by a parasite, such as malaria. Thedisease or disorder may be an autoimmune disease, such as acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcalnephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarterifis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, parnphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis. The disease or disorder may be myocardialinfarction, ischemic heart disease, or atherosclerotic plaques, or graftrejection, or Alzheimer's disease, or caused by atopic tissue. Thedisease or disorder may be inflammation caused by accretion of activatedgranulocytes, monocytes, lymphoid cells or macrophages at the site ofinflammation, and where the inflammation is caused by an infectiousagent.

Also provided is a pretargeting method of treating or diagnosing anon-neoplastic disease or disorder in a subject by (a) administering tothe subject the polyvalent protein complex of claim 1, where two antigenbinding sites are directed to a marker substance, or marker substancesspecific for the disorder, and one antigen binding sites is directed toa targetable construct containing a bivalent hapten; (b) optionallyadministering to the subject a clearing composition, and allowing thecomposition to clear the polyvalent complex from circulation; and (c)administering to the subject the targetable construct containing abivalent hapten, where the targetable construct further contains one ormore chelated or chemically bound therapeutic or diagnostic agents. Thedisease or disorder may be as described above.

Also provided is a method of antibody dependent enzyme prodrug therapy(ADEPT) by; a) administering to a patient with a neoplastic disorder thepolyvalent protein complex as above, where the complex contains acovalently attached enzyme capable of activating a prodrug, (b)optionally administering to the subject a clearing composition, andallowing the composition to clear the polyvalent complex fromcirculation, and (c) administering the prodrug to the patient.

Also provided are assay and immunostaining methods using one or morepolyvalent protein complexes as described above.

Further provided is an isolated nucleic acid molecule encoding a firstor second polypeptide as described above, and a nucleic acid expressioncassette containing such an isolated nucleic acid. Also provided is anepisome containing: (a) a first promoter operationally connected to afirst nucleic acid encoding a first polypeptide containing a polypeptidechain represented by the formula a₁-1₁-a₂-1₂-a₃, where a₁, a₂, and a₃are immunoglobulin variable domains and 1₁ and 1₂ are peptide linkers,(b) a second promoter operationally connected to a second nucleic acidencoding a polypeptide containing a second polypeptide chain representedby the formula b₁-1₃-b₂-14-b₃, where b₁, b₂, and b₃ are immunoglobulinvariable domains and 1₃ and 1₄ are peptide linkers, where the first andsecond polypeptide chain together form a complex containing at leastthree antigen binding sites, where each of the antigen binding sitescontains a variable domain from the first polypeptide chain and avariable domain from the second polypeptide chain, where the firstnucleic acid and the second nucleic acid are coexpressed when theepisome is transformed into a host cell. The episome may be a plasmid ora cosmid. Also provided is a host cell containing a nucleic acid, aexpression cassette and/or an episome as described above. The host cellmay be, for example, E. coli, yeast, a plant cell and a mammalian cell.

Also provided are methods of preparing a polyvalent protein complex,containing culturing a host cell as described above. The host cell maybe, for example, a murine myeloma cell line. The episome may contain theplasmid is pdHL2.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of preferred embodimentsof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Panel A shows an expression cassette in BS14HP-GAP+vector, whichcodes for a two species of mRNA synthesized from the constitutive GAPpromoters.

Panel B shows a drawings of the two mature heterologous polypeptides,h679V_(H)-GGGGS-hMN-14V_(K)-LEGGGS-hMN-14V_(H)-6His (Left) andhMN-14V_(K)-GGGQFM-hMN-14V_(H)-GGGGS-h679V_(K)-6His (Right), followingcleavage of the α factor signal peptides by Kex2 protease.

Panel C shows a drawing of a trivalent protein structure formed by theheterodimerization of polypeptides 1 and 2 possessing two binding sitesfor CEA and one for HSG.

Panel D shows the amino acid sequence and cDNA sequence ofEAEAEFM-h679VH-GGGGS-hMN-14VK-LEGGGS-hMN-14VH-6His.

Panel E shows the amino acid sequence and cDNA sequence ofEAEAEF-hMN-14VK-GGGQFM-hMN-14VH-GGGGS-h679VK-6His.

FIG. 2 depicts the construction of the modified Pichia expression vectorpGAPZα+ used for the co-expression of two heterologous polypeptides fromthe same host cell.

FIG. 3 shows one of many BIAcore sensorgrams used to evaluate expressionof BS14HP in the culture media of Pichia pastoris clones. Followinggrowth to stationary phase, culture media was diluted ten-fold inBIAcore eluent buffer and injected over an HSG-coupled sensorchip. Asubsequent injection of WI2 IgG (anti-id to hMN-14) confirmed thebispecificity of the samples.

FIG. 4 shows a Coomassie blue-stained SDS-PAGE gel of BS14HP. Purifiedprotein samples were subjected to reducing SDS-PAGE on 4-20%polyacrylamide Tris-Glycine gels. 1, 4 and 10 μg were loaded inindicated lanes. Arrows indicate the positions of molecular weightstandards.

FIG. 5 shows the size exclusion HPLC profile of purified BS14HP.

FIG. 6(a) shows a graphical representation of the results of acompetitive ELISA experiment. HRP-conjugated hMN14 IgG (1 nM) was mixedwith either BS14HP, BS1.5H (a bispecific diabody, monovalent for CEA andmonovalent for HSG, derived from the same variable domains as BS14HP) orhMN14 F(ab′)₂ at concentrations ranging from 1-250 nM, prior toincubation in CEA-coated (0.5 μg/well) wells. The % inhibition isplotted vs. nM concentration of sample. The 50% inhibitory concentration(IC₅₀) is given for each and (b) shows the results of SE-HPLC analysisof BS14HP immunoreactivity with CEA.

FIG. 7 shows a graphical representation of the tumor residence and bloodclearance of ¹²⁵I, labeled BS14HP in GW39tumor bearing nude mice. The %injected dose/gram (% ID/g) is plotted versus time (hours).

FIG. 8 shows the biodistribution (A) and tumor/non-tumor ratios after 3hours (B) of ¹¹¹In-IMP-241 in GW-39 tumor bearing mice pretargeted withthree bispecific constructs and (C) and tumor/non-tumor ratios after 24hours. Standard deviations are shown as error bars (A and C) or as ± inparentheses (B).

FIG. 9 panel A shows the molecular structure of IMP 281, panel B showsthe molecular structure if IMP 284, panel C shows the figure of IMP 288.

FIG. 10 depicts constructs of SV3 construct and ORF1 and ORF2polypeptide.

FIG. 11 is a schematic representation of hBS14-pDHL2 expression vector.

FIG. 12 depicts the results of MTX amplification of hBS14 SP2/0 clone1H6.

FIG. 13 depicts the results of SE-HPLC analysis of purified hBS14.

FIG. 14 depicts the results of SDS-PAGE analysis of purified hBS14.

FIG. 15 depicts the results of IEF analysis of purified hBS14.

FIG. 16 depicts the results of BIAcore analysis of hBS14.

FIG. 17 depicts the results of BIAcore analysis of HSG binding of hBS14produced in either SP2/0 or YB2/0 cells.

FIG. 18 shows the structure of the peptide IMP 291.

FIG. 19 shows the structure of the peptide IMP 245.

FIG. 20 shows the tumor uptake of ¹²⁵I-hBS14 and ^(99m)Tc-IMP-245 inmice when the hBS14 was given 4 hrs (top panel) or 24 hrs (bottom panel)to clear prior to administration of peptide (Groups I and IIrespectively).

FIG. 21 the top panel shows the tumor uptake of ¹²⁵I-hBS14 and^(99m)Tc-IMP-245 in mice given 48 hrs to clear the hBS114 prior theadministration of the peptide (Group III). The bottom panel showspeptide uptake in imaged mice at 24 hr post-injection.

FIG. 22 is a table showing percent ID/g and tumor/non-tumor ratios of^(99m)Tc-IMP-245 peptide at 1 h post injection.

FIG. 23 shows imaging data in mice. The first pair of images shows thelocation of the tumors in the mice. The second pair of images shows theimage at 1 hr post-peptide administration. The third pair of images showimaging data at 3 hrs post-peptide administration. The final pair ofimages shows the image at 24 hrs post-peptide administration.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

As used herein, the term “engineered antibody” encompasses allbiochemically or recombinately produced functional derivatives ofantibodies. A protein is a functional derivative of an antibody if ithas at least one antigen binding site (ABS) or acomplementarity-determining region (CDR) that when combined with otherCDR regions (on the same polypeptide chain or on a different polypeptidechain) can form an ABS. The definition of engineered antibody wouldinclude, at least, recombinant antibodies, tagged antibodies, labeledantibodies, Fv fragments, Fab fragments, recombinant (as opposed tonatural) multimeric antibodies, single chain antibodies, diabodies,triabodies, tetravalent multimers (dimer of diabodies), pentavalentmultimers (dimer of diabody and triabody), hexavalent multimers (dimerof triabodies) and other higher multimeric forms of antibodies.

As used herein, the term “single-chain antibody (scFv),” refers toengineered antibody constructs prepared by isolating the binding domains(both heavy and light chain) of a binding antibody, and supplying alinking moiety which permits preservation of the binding function. Thisforms, in essence, a radically abbreviated antibody, having only thevariable domain necessary for binding the antigen. Determination andconstruction of single chain antibodies are described in many priorpublications including U.S. Pat. No. 4,946,778; Bird et al., Science242:423 (1988) and Huston et al., Proc. Nat'l Acad. Sci. USA85:5879(1988).

The term “humanized” means that at least a portion of the frameworkregions of an immunoglobulin or engineered antibody construct (includingthe PPC of this invention that comprise an immunoglobulin or engineeredantibody) is derived from human immunoglobulin sequences. It should beclear that any method to humanize antibodies or antibody constructs, asfor example by variable domain resurfacing as described by Roguska etal., (1994) Proc. Natl. Acad. Sci. USA 91: 969-973 would be applicableto the PPC of this invention. Alternatively, CDR grafting (also calledCDR shuffling) or reshaping as reviewed by Hurle and Gross ((1994) Curr.Opin. Biotech. 5:428-433), can be used. Manipulation of thecomplementarity-determining regions (CDR) is a way of achievinghumanized antibodies. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. See, for example,U.S. Pat. Nos. 5,874,540 and 6,254,868. General techniques for cloningmurine immunoglobulin variable domains are described, for example, bythe publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833(1989). Techniques for producing humanized MAbs are described, forexample, by Jones et al., Nature 321: 522 (1986), Riechmann et al.,Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit.Rev. Biotech. 12: 437 (1992), Singer et al., J. Immun. 150: 2844 (1993),Winter & Milstein, Nature 349:293 (1991).

The terms “recombinant nucleic acid” or “recombinantly produced nucleicacid” refer to nucleic acids such as DNA or RNA which has been isolatedfrom its native or endogenous source and modified either chemically orenzymatically by adding, deleting or altering naturally-occurringflanking or internal nucleotides. Flanking nucleotides are thosenucleotides which are either upstream or downstream from the describedsequence or sub-sequence of nucleotides, while internal nucleotides arethose nucleotides which occur within the described sequence orsubsequence.

The term “recombinant means” refers to techniques where proteins areisolated, the cDNA sequence coding the protein identified and insertedinto an expression vector. The vector is then introduced into a cell andthe cell expresses the protein. Recombinant means also encompasses theligation of coding or promoter DNA from different sources into onevector for expression of a PPC, constitutive expression of a protein, orinducible expression of a protein.

The term “promoter” refers to a DNA sequence which directs thetranscription of a structural gene to produce mRNA. Typically, apromoter is located in the 5′ region of a gene, proximal to the startcodon of a structural gene. If a promoter is an inducible promoter, thenthe rate of transcription increases in response to an inducing agent. Incontrast, the rate of transcription is not regulated by an inducingagent if the promoter is a constitutive promoter.

The term “enhancer” refers to a promoter element. An enhancer canincrease the efficiency with which a particular gene is transcribed intomRNA irrespective of the distance or orientation of the enhancerrelative to the start site of transcription.

“Complementary DNA (cDNA)” refers to a single-stranded DNA molecule thatis formed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complement.

“Expression” refers to the process by which a polypeptide is producedfrom a structural gene. The process involves transcription of the geneinto mRNA and the translation of such mRNA into polypeptide(s).

“Cloning vector” refers to a DNA molecule, such as a plasmid, cosmid,phagemid, or bacteriophage, which has the capability of replicatingautonomously in a host cell and which is used to transform cells forgene manipulation. Cloning vectors typically contain one or a smallnumber of restriction endonuclease recognition sites at which foreignDNA sequences may be inserted in a determinable fashion without loss ofan essential biological function of the vector, as well as a marker genewhich is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

“Expression vector” refers to a DNA molecule comprising a clonedstructural gene encoding a foreign protein which provides the expressionof the foreign protein in a recombinant host. Typically, the expressionof the cloned gene is placed under the control of (i.e., operably linkedto) certain regulatory sequences such as promoter and enhancersequences. Promoter sequences may be either constitutive or inducible.

“Recombinant Host” or “Host cell” refers to a prokaryotic or eukaryoticcell which contains either a cloning vector or expression vector. Thisterm is also meant to include those prokaryotic or eukaryotic cells thathave been genetically engineered to contain the cloned gene(s) in thechromosome or genome of the host cell. The host cell is not limited to aunicellular organism such as E. coli and yeast. Cells from multicellularorganisms such as mammals, insects, and plants are also contemplated ashost cells in the context of this invention. For examples of suitablehosts, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989). A mammalian host cell may be of any mammalian origin andinclude, at least cells of human, bovine, canine, murine, rattus,equine, porcine, feline and non-human primate origin.

A “tumor-associated antigen” is a protein normally not expressed, orexpressed at very low levels, by a normal cell. However, in a neoplasticor preneoplastic cell (a cell predisposed to becoming a cancer cell),the tumor-associated antigen is expressed at a level that is higher thanthat of a normal cell. The preferred tumor-associated antigens are theones that are expressed at very high levels in neoplastic andpreneoplastic cells but at very low levels or not expressed in normalcells. “Antigens” and “tumor-associated antigens” are well known andinclude at least α-fetoprotein, A3, A33 (GI cancers, particularly coloncancer), CA125, carcinoembryonic antigen (CEA), CD19, CD20, CD21, CD22,CD23, CD30, CD33, CD45, CD52 (associated with chronic lymphocyticleukemia and other lymphomas), CD74, CD66, CD80, colon-specificantigen-p (CSAp), EGFR, EGP-1, EGP-2, folate receptor, HER2/neu, HLA-DR,human chorionic gonadrotropin, Ia, IL-2, IL-6 (prostate cancer),insulin-like growth factor, KS-1, Le(y), MAGE, MUC1, MUC2, MUC3, MUC4,necrosis antigens, PAM-4, placental growth factor, prostatic acidphosphatase (PAP), prostate specific antigen (PSA), PSMA, S100, T101,TAC, TAG-72, tenascin, and VEGF. Furthermore, the ABS of the inventionincludes, at least, an ABS that binds to an epitope of the above listedantigens. Tumor-associated antigens may be either produced by the tumorcells themselves or by adjacent structures, such as the tumor's vascularendothelium. B-cell, T-cell and other such “lineage” antigens which arepresent in both normal and malignant cell types may still be usefultargets because of a differential expression by or sensitivity of themalignant cells to antibodies against these lineage antigens (e.g.,CD19, CD20, CD21, CD22 in normal and malignant B cells). Many otherillustrations of tumor-associated antigens are known to those of skillin the art. See, e.g., Urban et al., Ann. Rev. Immunol. 10:617 (1992).The list above is illustrative only and cites the cancers most closelyassociated to the tumor-associated antigen. In most cases, eachtumor-associated antigen may have up to 2, 3, 4, 5, 6 or more epitopes.

Known tumors that are associated with tumor-associated antigens include,at least, carcinomas, melanomas, sarcomas, gliomas, myelomas, leukemiasand lymphomas.

As used herein, an “infectious agent” and “pathogen” denotes bothmicrobes and parasites. A “microbe” includes viruses, bacteria,rickettsia, mycoplasma, protozoa, fungi and like microorganisms. A“parasite” denotes infectious, generally microscopic or very smallmulticellular invertebrates, or ova or juvenile forms thereof, which aresusceptible to antibody-induced clearance or lytic or phagocyticdestruction, such as malarial parasites, spirochetes, and the like.Examples of infectious agents include, for example, a fungus, virus,parasite, bacterium, protozoan, or mycoplasm. The fungus may be from thespecies of Microsporum, Trichophyton, Epidermophyton, Ssporothrixschenckii, Cyrptococcus neoformans, Coccidioides immitis, Histoplasmacapsulatum, Blastomyces dermatitidis, or Candida albicans. The virus maybe from the species of human immunodeficiency virus (HIV), herpes virus,cytomegalovirus, rabies virus, influenza virus, hepatitis B virus,Sendai virus, feline leukemia virus, Reo virus, polio virus, human serumparvo-like virus, simian virus 40, respiratory syncytial virus, mousemammary tumor virus, Varicella-Zoster virus, Dengue virus, rubellavirus, measles virus, adenovirus, human T-cell leukemia viruses,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus,wart virus and blue tongue virus. The bacterium may be, for example,Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis andTetanus toxin. The parasite may be a helminth or a malarial parasite.The protozoan may be Plasmodium falciparum, Plasmodium vivax, Toxoplasmagondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosomarhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosomajapanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus,Leishmania tropica, Trichinella spiralis, Onchocerca volvulus, Theileriaparva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus or Mesocestoides corti. The mycoplasma may be Mycoplasmaarthritidis, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasmaarginini, Acholeplasma laidlawii, Mycoplasma salivarum, and Mycoplasmapneumoniae. Other examples of infectious agents and pathogens that maybe treated by the product (PPC) and methods of this invention arecontained in the second and subsequent editions of Davis et al,“Microbiology” (Harper & Row, New York, 1973 and later), and are wellknown to the ordinary skilled art worker.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing, curing, reversing, attenuating,alleviating, minimizing, suppressing or halting the deleterious effectsof a disease state, disease progression, disease causative agent (e.g.,bacteria or viruses) or other abnormal condition. Because some of theinventive methods involve the physical removal of the etiological agent,the artisan will recognize that they are equally effective in situationswhere the inventive compound is administered prior to, or simultaneouswith, exposure to the etiological agent (prophylactic treatment) andsituations where the inventive compounds are administered after (evenwell after) exposure to the etiological agent.

Unless otherwise noted, use of the term “antibody” or “immunoglobulin”herein will be understood to include antibody fragments and functionalderivatives (i.e., engineered antibody) thereof. Antibodies can be wholeimmunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE, or hybridantibodies with dual or multiple antigen or epitope specificities, orfragments, e.g., F(ab′)₂, F(ab)₂, Fab′, Fab₁ and the like, includinghybrid fragments. Functional derivatives include engineered antibodies.

The terms “recombinant protein,” “recombinantly produced protein” or“recombinantly produced immunotoxin” refer to a peptide or proteinproduced using non-native cells that do not have an endogenous copy ofDNA able to express the protein. The cells produce the protein becausethey have been genetically altered by the introduction of theappropriate nucleic acid sequence. The recombinant protein will not befound in association with proteins and other subcellular componentsnormally associated with the cells producing the protein.

The term “selective cytotoxic reagent” refers to a compound that whenadded to a population of different cells, e.g., within an organism,kills one type of cell in the population based on some physicalcharacteristic of the cell, i.e., a surface ligand or marker to whichthe cytotoxic reagent binds and then becomes internalized.

The term “surface marker” refers to various constituents, such as aprotein, carbohydrate, or glycoprotein, that are present on the surfaceof a cell. Different types of cells express different cell surfacemarkers and therefore cells can be identified by the presence of a cellsurface marker. For example, B cells express CD19, CD20 (See, Ansell etal., J. Clin. Oncology, 20:3885-3890 (2002) and Witzig et al., J. Clin.Oncology 20:2453-2463) and CD22. Thus, the binding of an antibody thatrecognizes CD19, CD20 or CD22 identifies that cell as a B cell, eithernormal or malignant. As another example, the B-cell may be a multiplemyeloma, in which case the B cells may express the tumor-associatedantigen MUC1 or CD74. B cell surface markers may be used for ablation ofB cells and B cell tumor-associated antigens may be used to ablate Bcell tumors such as the multiple myeloma described above.

The term “CD22” refers to a lineage-restricted B-cell antigen belongingto the Ig superfamily, is expressed on the surface of many types ofmalignant B cells, including but not limited to, acute lymphocyticleukemia (B-ALL), chronic B-lymphocytic cells (B-CLL), B lymphoma cellssuch as Burkitt's, AIDS-associated and follicular lymphomas, and hairycell leukemias, as well as on normal mature B lymphocytes. See, U.S.Pat. Nos. 6,183,744 and 6,306,393. CD22 is not expressed in early stagesof B-cell development, nor is it found on the surface of stem cells orterminal stage plasma cells. Vaickus et al., Crit. Rev. Oncol/Hematol.11:267-297 (1991). Additionally, no shed antigen is detected in normalhuman serum or serum from patients with CLL. Li et al., Cell. Immunol.118:85-99 (1989).

According to the specific case, the “therapeutically effective amount”of an agent should be determined as being the amount sufficient toimprove the symptoms of the patient in need of treatment or at least topartially arrest the disease and its complications. Amounts effectivefor such use will depend on the severity of the disease and the generalstate of the patient's health. Single or multiple administrations may berequired depending on the dosage and frequency as required and toleratedby the patient.

As used herein, the term “a method to detect” refers to any assay(including immunoassays and colorimetric assays) known in the art forthe measurement of a detectable label. These assays include, at least,assays utilizing biotin and avidin (including streptavidin), ELISA's andimmunoprecipitation, immunohistochemical techniques and agglutinationassays. A detailed description of these assays is given in WO 96/13590to Maertens & Stuyver. The term “biological sample” relates to anypossible sample taken from an animal (including humans), such as blood(which also encompasses serum and plasma samples), sputum, cerebrospinalfluid, urine, lymph or any possible histological section, and other bodyfluid. Detection may also include methods of imaging a lesion, such aswith immunoscintigraphy, computed tomography (CT), ultrasonography,X-rays, and the like.

The terms “binding specificity,” “specifically binds to” or“specifically immunoreactive with,” when referring to a protein or ABSof the invention, refers to a binding reaction which is determinative ofthe presence of the protein or carbohydrate in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified PPC bind to aparticular protein or carbohydrate and do not bind in a significantamount to other proteins or carbohydrates present in the sample.Specific binding to a PPC under such conditions may require a PPCselected for its specificity towards a particular protein orcarbohydrate. For example, PPCs specific for the CD22 antigen may beselected to provide PPC that are specifically immunoreactive with CD22protein and not with other proteins. A variety of immunoassay formatsmay be used to select PPC specifically immunoreactive with a particularprotein or carbohydrate. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein or carbohydrate. See Harlow & Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Publication, New York (1988) for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

The terms “isolated” or “substantially purified,” when applied to anucleic acid or protein, denotes that the nucleic acid or protein isessentially free of other cellular components with which it isassociated in the natural state. It is preferably in a homogeneousstate, although it can be in either a dry or aqueous solution. Purityand homogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein which is the predominantspecies present in a preparation is substantially purified.

The terms “nucleic acid encoding” or “nucleic acid sequence encoding”refer to a nucleic acid which directs the expression of a specificprotein or peptide. The nucleic acid sequences include both the DNAstrand sequence that is transcribed into RNA and the RNA sequence thatis translated into protein. The nucleic acid sequences include both fulllength nucleic acid sequences as well as shorter sequences derived fromthe full length sequences. It is understood that a particular nucleicacid sequence includes the degenerate codons of the native sequence orsequences which may be introduced to provide codon preference in aspecific host cell. The nucleic acid includes both the sense andantisense strands as either individual single strands or in the duplexform.

“Pharmaceutical composition” refers to formulations of variouspreparations. Parenteral formulations are known and are preferred foruse in the invention. The formulations containing therapeuticallyeffective amounts of the immunotoxins are either sterile liquidsolutions, liquid suspensions or lyophilized versions and optionallycontain stabilizers or excipients. Lyophilized compositions arereconstituted with suitable diluents, e.g., water for injection, saline,0.3% glycine and the like, at a level of about from 0.01 mg/kg of hostbody weight to 10 mg/kg or more.

The term “crosslinker” is well known in the art and include at leastABH(21509), AEDP(22101), AMAS(22295), ANB-NOS(21451), APDP(27720),APG(20108), ASBA(21512), BASED(21564), BMB(22331), BMDB(22332),BMH(22330), BMOE(22323), BMPA(22296), BMPH(22297), BMPS(22298),BM[PEO]₃(22336), BM[PEO]₄(22337), BSOCOES(21600), BS3(21580),DFDNB(21525), DMA(20663), DMP(21666), DMS(20700), DPDPB(21702),DSG(20593), DSP(22585), DSS(21555), DST(20589), DTBP(20665),DTME(22335), DTSSP(21578), EDC(22980), EGS(21565), EMCA(22306),EMCH(22106), EMCS(22308), GMBS(22309), HBVS(22334), KMUA(22211),KMUH(22111), LC-SMCC(22362), LC-SPDP(21651), MBS(22311), M2C2H(22303),MPBH(22305), MSA(22605), NHS-ASA(27714), PDPH(22301), PMPI(28100),SADP(21533), SAED(33030), SAND(21549), SANPAH(22600), SASD(27716),SATA(26102), SATP(26100), SBAP(22339), SFAD(27719), SIA(22349),SIAB(22329), SMCC(22360), SMPB(22416), SMPH(22363), SMPT(21558),SPDP(21857), Sulfo-BSOCOES(21556), Sulfo-DST(20591), Sulfo-EGS(21566),Sulfo-EMCS(22307), Sulfo-GMBS(22324), Sulfo-HSAB(21563),Sulfo-KMUS(21111), Sulfo-LC-SPDP(21650), Sulfo-MBS(22312),Sulfo-NHS-LC-ASA(27735), Sulfo-SADP(21553), Sulfo-SANPAH(22589),Sulfo-SIAB(22327), Sulfo-SMCC(22322), Sulfo-SMPB(22317),Sulfo-LC-SMPT(21568), Sulfo-SBED(33033), SVSB(22358), TFCS(22299),THPP(22607), TMEA(33043), and TSAT(33063) (Pierce Chemical, Rockford,Ill. catalog number in parenthesis). See, also U.S. Pat. No. 4,680,338and provisional patent application 60/436,359 filed Dec. 24, 2002, foradditional linker descriptions.

The term “chemotherapeutic agent” may be any chemotherapeutic agentknown in the art and includes, at least, taxanes, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes;folic acid analogs, pyrimidine analogs, purine analogs, vinca alkaloids,antibiotics, enzymes, platinum coordination complexes, substituted urea,methyl hydrazine derivatives, adrenocortical suppressants, orantagonists. Specifically, the chemotherapeutic agent may be from thegroup of steroids, progestins, estrogens, antiestrogens, and androgens.More specifically, the chemotherapeutic agent may be azaribine,bleomycin, bryostatin-1, busulfan, carmustine, celebrex, chlorambucil,cisplatin, CPT-11, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, ethinyl estradiol, etoposide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,methotrexate, methotrexate, mithramycin, mitomycin, mitotane, phenylbutyrate, prednisone, procarbazine, semustine streptozocin, tamoxifen,taxanes, taxol, testosterone propionate, thalidomide, thioguanine,thiotepa, uracil mustard, vinblastine, and vincristine.

The term “cytotoxic agents” includes all known cytotoxic and cytostaticagents. Examples of these agents are listed in Goodman et al., “THEPHARMACOLOGICAL BASIS OF THERAPEUTICS,” Sixth Edition, A. G. Gilman etal, eds./Macmillan Publishing Co. New York, 1980, as well as a morecurrent edition (See also, U.S. Pat. Nos. 6,083,477 and 6,395,276) Theseagents include, at least the following: antiapoptotic agents,antimetabolites, alkaloids, antimitotic agents, enzyme inhibitors,COX-inhibitors, chemotherapeutic agents; antibiotics, such asdactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin andmitomycin; enzymes, such as L-asparaginase; platinum coordinationcomplexes, such as cisplatin; substituted urea, such as hydroxyurea;methyl hydrazine derivatives, such as procarbazine; adrenocorticalsuppressants, such as mitotane; hormones and antagonists, such asadrenocortisteroids (prednisone), progestins (hydroxyprogesteronecaproate, medroprogesterone acetate and megestrol acetate), estrogens(diethylstilbestrol and ethinyl estradiol), antiestrogens (tamoxifen),and androgens (testosterone propionate and fluoxymesterone). Otherexamples of cytotoxic agents include ricin, abrin, ribonuclease, DNaseI, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin andradionuclides. See, for example, Pastan et al., Cell 47:641 (1986), andGoldenberg, CA—A Cancer Journal for Clinicians 44:43 (1994). Othersuitable toxins are known to those of skill in the art.

Therapeutic agents are as defined in the specification but include atleast, an immunoe modulator, an enzyme, a hormone.

Radionuclide include any radioactive isotope useful for medicaldiagnostic, therapeutic and imaging methods (i.e., detectable labels).Examples of radionuclides include ²²⁵Ac, ¹¹¹Ag, ⁷²As, ⁷⁷As, ²¹¹At,¹⁹⁸Au, ¹⁹⁹Au, ²¹²Bi, ²¹³Bi, ⁷⁵Br, ⁷⁶Br, ¹¹C, ⁵⁵Co, ⁶²Cu, ⁶⁷Cu, ¹⁶⁶Dy,¹⁶⁹Er, ¹⁸F, ⁵²Fe, ⁵⁹Fe, ⁶⁷Ga, ⁶⁸Ga, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁶⁶Ho, ¹²⁰I, ¹²¹I, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹⁰In, ¹¹¹In, ¹⁹⁴Ir, ¹⁷⁷Lu, ⁵¹Mn, ⁹⁹Mn, ⁹⁹Mo, ¹³N,¹⁵O, ³²P, ³³P, ²¹¹Pb, ²¹²Pb, ¹⁰⁹Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁴³Pr, ²²³Ra,^(82m)Rb, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁰⁵Rh, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ⁸³Sr, ⁸⁹Sr,¹⁶¹Tb, ^(94m)Tc, ^(99m)Tc, ⁸⁶Y, ⁹⁰Y and ⁸⁹Zr. Of these radionuclides²²⁵Ac, ¹¹¹Ag, ⁷⁷As, ²¹¹At, ¹⁹⁸Au, ¹⁹⁹Au, ²¹²Bi, ²¹³Bi, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,¹⁶⁶Dy, ¹⁶⁹Er, ⁵⁹Fe, ⁶⁷Ga, ¹⁶⁶Ho, ¹²⁵I, ¹³¹I, ¹¹¹In, ¹⁹⁴Ir, ¹⁷⁷Lu, ⁹⁹Mo,³²P, ³³P, ²¹¹Pb, ²¹²Pb, ¹⁰⁹Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁴³Pr, ²²³Ra, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁰⁵Rh, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ⁸⁹Sr, ¹⁶¹Tb and ⁹⁰Y are particularlyuseful as therapeutic radionuclides and therapeutic cations. Further,⁷²As, ⁷⁵Br, ⁷⁶Br, ¹¹C, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸F, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga,¹⁵⁴⁻¹⁵⁸Gd, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ⁵¹Mn,⁵²Mn, ¹³N, ¹⁵O, ³²P, ²²³Ra, ⁸²Rb, ¹⁸⁶Re, ¹⁸⁸Re, ⁸³Sr, ⁹⁴Tc, ⁹⁹Tc, ⁸⁶Y,⁹⁰Y and ⁸⁹Zr are particularly useful as diagnostic radionuclides anddiagnostic cations.

“Antimicrobial agents” are any agents that has a cytotoxic or cytostaticeffect on microbes. Antimicrobial agents may be conventionallyclassified into four main groups, based upon their affecting (1)bacterial cell-wall synthesis, (2) the cytoplasmic membrane, (3) proteinsynthesis, and (4) nucleic acid synthesis, and often each of thesegroups can be subdivided into several classes. Reviews of antimicrobialchemotherapy can be found in the chapter by M. P. E. Slack (In: OxfordTextbook of Medicine, Second Ed., Vol. 1, edited by D. J. Weatherall, J.G. G. Lidingham, and D. A. Warrell, pp. 5.35-5.53; Oxford UniversityPress, Oxford/Melbourne/New York, 1987) and in Section XII, Chemotherapyof Microbial Diseases (In: Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 6th Ed., Goodman et al., Eds., pp. 1080-1248;Macmillan Publishing Co., New York, 1980—and also the 2001 edition). Asindicated in these texts, some antimicrobial agents are selective intheir toxicity, since they kill or inhibit the microorganism atconcentrations that are tolerated by the host (i.e., the drug acts onmicrobial structures or biosynthetic pathways that differ from those ofthe host's cells). Other agents are only capable of temporarilyinhibiting the growth of the microbe, which may resume growth when theinhibitor is removed. Often, the ability to kill or inhibit a microbe orparasite is a function of the agent's concentration in the body and itsfluids.

Cytokines are known to those of skill in the art and includes, at least,“immune modulators” such as IL-1, IL-2, IL-3, IL-6, IL-1, IL-12, IL-18,IL-21, interferon-α, interferon-β, and interferon-γ.

It is understood that the definitions provided above are not mutuallyexclusive. For example, one molecule may be a cytotoxic agent, aradionuclide and a detectable label.

Structure of the Polyvalent Protein Complex (PPC)

The invention provides for a polyvalent protein complex (PPC) comprisingtwo polypeptide chains generally arranged laterally to one another. Eachpolypeptide chain typically comprises 3 or 4 “v-regions”, which compriseamino acid sequences capable of forming an antigen binding site whenmatched with a corresponding v-region on the opposite polypeptide chain.Up to about 6 “v-regions” can be used on each polypeptide chain,however. The v-regions of each polypeptide chain are connected linearlyto one another and may be connected by interspersed linking regions.When arranged in the form of the PPC, the v-regions on each polypeptidechain form individual antigen binding sites. Thus, for example, a PPCwith 4 antigen binding sites and three linking regions can be depictedas follows:

-   -   [amino terminus]-a₁-1-a₂-1₂-a₃-1₃-a₄-[carboxyl terminus]    -   [carboxyl terminus]-b₁-1₄-b₂-1₅-b₃-1₆-b₄-[amino terminus]

As shown here, the first polypeptide comprises 4 v-regions, a₁, a₂, a₃and a₄, connected by three linker regions, 1₁, 1₂ and 1₃. The secondpolypeptide of the PPC comprises 4 corresponding v-regions b₁, b₂, b₃and b₄ and three interspersed linker regions, 1₄, 1₅ and 1₆. Theindividual polypeptide chains of the PPC are bound to one another by thecomplementarity binding of the corresponding v-regions on each chain.Thus, as depicted above, a₁ binds to b₁, a₂ binds to b₂, a₃ binds to b₃,etc. to form the PPC.

The PPC of the invention can comprise v-regions of various amino acidsequences so long as the arrangement of corresponding v-regions on thetwo polypeptide chains (i.e., a_(n) to b_(n)) provides for an antigenbinding site. The binding of the corresponding v-regions forms theindividual antigen binding sites of the PPC. A preferred method forforming each antigen binding site on the PPC is to arrange correspondingV_(H) and V_(L) regions of known antigen binding regions from antibodiesor antibody fragments. However the practice of the invention is notlimited to incorporation of such known antigen binding regions. Ifcorresponding V_(H) and V_(L) regions are used, there are no limitationson which of the two v-regions (i.e., a_(n) or b_(n)) encode V_(H) orV_(L). For example, where n=3, any combination of V_(H) and V_(L) listedbelow are possible: Combination a₁ a₂ a₃ b₁ b₂ b₃ 1 V_(H) V_(H) V_(H)V_(L) V_(L) V_(L) 2 V_(H) V_(H) V_(L) V_(L) V_(L) V_(H) 3 V_(H) V_(L)V_(H) V_(L) V_(H) V_(L) 4 V_(L) V_(H) V_(H) V_(H) V_(L) V_(L) 5 V_(H)V_(L) V_(L) V_(L) V_(H) V_(H) 6 V_(L) V_(H) V_(L) V_(H) V_(L) V_(H) 7V_(L) V_(L) V_(H) V_(H) V_(H) V_(L) 8 V_(L) V_(L) V_(L) V_(H) V_(H)V_(H)

As a further example, in the case where there are four V-regions, any ofthe following are possible. Combination a₁ a₂ a₃ a₄ b₁ b₂ b₃ b₄ 1 V_(H)V_(H) V_(H) V_(H) V_(L) V_(L) V_(L) V_(L) 2 V_(H) V_(H) V_(L) V_(H)V_(L) V_(L) V_(H) V_(L) 3 V_(H) V_(L) V_(H) V_(H) V_(L) V_(H) V_(L)V_(L) 4 V_(L) V_(H) V_(H) V_(H) V_(H) V_(L) V_(L) V_(L) 5 V_(H) V_(L)V_(L) V_(H) V_(L) V_(H) V_(H) V_(L) 6 V_(L) V_(H) V_(L) V_(H) V_(H)V_(L) V_(H) V_(L) 7 V_(L) V_(L) V_(H) V_(H) V_(H) V_(H) V_(L) V_(L) 8V_(L) V_(L) V_(L) V_(H) V_(H) V_(H) V_(H) V_(L) 9 V_(H) V_(H) V_(H)V_(L) V_(L) V_(L) V_(L) V_(H) 10 V_(H) V_(H) V_(L) V_(L) V_(L) V_(L)V_(H) V_(H) 11 V_(H) V_(L) V_(H) V_(L) V_(L) V_(H) V_(L) V_(H) 12 V_(L)V_(H) V_(H) V_(L) V_(H) V_(L) V_(L) V_(H) 13 V_(H) V_(L) V_(L) V_(L)V_(L) V_(H) V_(H) V_(H) 14 V_(L) V_(H) V_(L) V_(L) V_(H) V_(L) V_(H)V_(H) 15 V_(L) V_(L) V_(H) V_(L) V_(H) V_(H) V_(L) V_(H) 16 V_(L) V_(L)V_(L) V_(L) V_(H) V_(H) V_(H) V_(H)

In one embodiment, one polypeptide of the PPC may be SEQ ID NO:1 (FIG.1D). In another embodiment, one polypeptide of the PPC may be SEQ IDNO:2 (FIG. 1E). In a preferred embodiment, one polypeptide of the PPC isSEQ ID NO:1 while the other polypeptide is SEQ ID NO:2.

Because each of the v-regions of the polypeptides of a PPC areindependent, each of the antigen binding sites can independently havethe same or different affinity or specificity. In separately preferredembodiments, the antigen binding sites of a PPC bind different epitopesor the same epitope. In the practice of this invention, in either suchembodiment, it is likely and acceptable that binding affinity for eachindividual antigen binding site will differ.

As noted above, a preferred embodiment of the PPC of this inventioncomprises known V_(H) and V_(L) sequences for the v-regions. Forexample, if it is desired for a PPC to have an ABS with the samespecificity as a target antibody. The gene for the target antibody maybe cloned or the target antibody may be subjected to protein sequencing.Then the V_(H) and V_(L) sequence of the target antibody may bedetermined. A nucleic acid construct may be made to coexpress bothpolypeptides of the PPC in a host where at least one of the PPC'santigen binding sites would comprise the corresponding V_(H) and V_(L)regions as the target antibody. These antigen binding sites would beexpected to have similar, if not identical, antigen binding specificityand affinity with the target antibody. In the practice of thisembodiment of the invention, the target antibody may be human, nonhumanor an engineered antibody. Furthermore, the antibody may be any antibodywhose sequence is in the public domain.

Methods of producing a target antibody of any specificity are known inthe art. For example, a monoclonal antibody may be made from an antigen.Recombinant antibody libraries expression libraries, which express arepertoire of antibodies on different host cells may be screened.Furthermore, antibodies may be purified and their protein sequencesdetermined using antigen affinity columns.

In another embodiment of the invention, the V_(H) and V_(L) regions ofthe PPC may be derived from a “humanized” monoclonal antibody or from ahuman antibody. Alternatively, the V_(H) and/or V_(L) regions maycomprise a sequence derived from human antibody fragments isolated froma combinatorial immunoglobulin library. See, for example, Barbas et al.,METHODS: A companion to Methods in Enzymology 2: 119 (1991), and Winteret al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expressionvectors that are useful for producing a human immunoglobulin phagelibrary can be obtained, for example, from STRATAGENE Cloning Systems(La Jolla, Calif.).

The human antibody V_(H) or V_(L) sequence may be derived from a humanmonoclonal antibody produced in a mouse. Such antibodies are obtainedfrom transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7: 13 (1994), Lonberg etal., Nature 368: 856 (1994), and Taylor et al., Int. Immun. 6: 579(1994).

The linker regions may comprise any amino acid sequence that are ofsufficient length to allow for arrangement of corresponding v-regions onthe individual polypeptide chains of the PPC into antigen binding sites(i.e. a₁/b₁, b₂/a₂, etc.), for example, due to steric constraints.However, the linker sequences should not be so long as to allow twoadjacent v-regions on the polypeptide chains to fold back on one another(i.e., a₁/a₂, b₁/b₂, etc.). Typically, linkers longer than 10 aminoacids are more likely to demonstrate folding back problems. In apreferred embodiment, the linkers comprise a polypeptide of between 3 to8 amino acids in length. While any amino acid may be used in the linker,the preferred amino acids are those that are flexible and hydrophilic(e.g., glycine and serine). Examples of such linkers include, forexample, the linkers of the invention as shown in FIGS. 1D and 1E. Insome embodiments where steric hindrance is not a constraint, the linkerregions may be omitted.

Tagged PPC

PPCs of the present invention may also be modified in a way to formchimeric molecules (referred to herein as “tagged PPC”) comprising afusion of a PPC with a “epitope tag” which provides an epitope to whichan anti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino or carboxyl terminus of the target protein.Provision of the epitope tag enables the target protein to be readilydetected, as well as readily purified by affinity purification. Varioustag epitopes are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 (see, Field et al. (1988) Mol. Cell.Biol. 8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Elnhaueret al., J. Biochem. Biophys. Methods, 2001 Oct. 30, 49(1-3), 455-65;Song et al., Int. J. Oncol. 2003 Jan., 22(1)₉₃-8; Werkmeister et al.,Biochim. Biophys Acta 1993 May 7, 1157(1): 50-4; Hopp et al. (1988)BioTechnology 6:1204); the KT3 epitope peptide (see, Martine et al.(1992) Science, 255:192); tubulin epitope peptide (see, Skinner (1991)J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag (see,Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.). Itis understood that tagged PPC is a subset of all PPC and any referenceto PPC in this disclosure also comprise tagged PPC.

In one embodiment of the invention, the three or four ABS of a PPC maybe specific for an epitope of a tumor-associated antigen. Each ABS of aPPC may be specific for a different tumor-associated antigen. Forexample, one tumor-associated antigen may be CEA while another tumorassociate antigen may be a non-CEA antigen. In another embodiment of theinvention, the PPC has at least one ABS specific for an epitope of ahapten. The hapten may be, for example, histamine-succinyl-glycine(HSG).

In another embodiment of the invention, the PPC is linked, via achemical bond, to a second molecule. These linkages may be made using acrosslinker. Alternatively, the linkage may be a binding pair such asantigen-antibody, hormone-receptor, drug-receptor, cell surfaceantigen-lectin, biotin-avidin, substrate/enzyme, peptide-receptor, andcomplementary nucleic acid strands, hapten-anti-hapten systems and thelike. The avidin described includes reduced affinity avidin and reducedimmunogenicity avidin as described by U.S. Pat. No. 5,698,405.

In one embodiment, the PPC may be linked to peptides (which includesproteins) to form a fusion PPC. The linkage may be any linkage thatcould be used to join two peptides since the PPC is itself comprised ofpeptides. For example, one method would be to synthesize the fusion PPCin a peptide synthesizer. In this case, the bond would be a peptide bond(also referred to as an amide bond). Another method would be tosynthesize or clone a DNA to encode both polypeptides of the fusion PPC.The DNA is placed into an expression vector and transformed into a hostcell permanently or transiently. Yet another method would be to use achemical crosslinker to join two peptides.

The PPC molecule of the invention may further comprise a “detectablelabel” such as a “diagnostic agent.” Detectable labels and diagnosticagents may include radiolabels, fluorescent labels, luminescent(chemiluminescent and bioluminescent) labels, positron-emissiontomography (PET) labels and SPECT labels. The choice of labels are wellknown but specific examples are provided below. Methods of detectinglabels are generally known and are also described in U.S. Pat. Nos.4,595,654, 4,735,210, 4,792,521, 5,364,612, 5,439,665, 5,632,968,5,697,902, 5,753,206, 6,071,490, 6,120,768, 6,126,916, and 6,187,284.The discussion of various labels in this segment of the disclosure isapplicable to all references to labels in this invention.

Radiolabels may be further classified as therapeutic cations anddiagnostic cations. Diagnostic cations may emit particles and/orpositrons having 25-10,000 keV. Therapeutic cations may emit particlesand/or positrons having 20 to 10,000 keV. Any conventional method ofradiolabeling which is suitable for labeling proteins for in vivo usewill be generally suitable for labeling the PPC of the invention. Suchmethods are known to the ordinary skilled artisan and are disclosedinter alia in, e.g., Childs et al., J. Nucl. Med., 26:293 (1985); and inU.S. Pat. Nos. 4,331,647,4,348,376, 4,361,544, 4,468,457, 4,444,744,4,624,846, 5,334,708, 5670,132, 5,514,363, 5,976,492, 6,358,489, and6,440,386. A wide range of labeling techniques are disclosed in Feteanu,“LABELED ANTIBODIES IN BIOLOGY AND MEDICINE”, pages 214-309 (McGraw-HillInt. Book Co., New York et al, 1978). The introduction of various metalradioiosotopes may be accomplished according to the procedures of Wagneret al., J. Nucl. Med., 20,428 (1979); Sundberg et al, J. Med. Chem., 17,1304 (1974); and Saha et al. J. Nucl. Med., 6, 542 (1976). Some of thesemethods describe the use of labeled antibodies. The methods may be usedin the present invention by the substitution of PPC of the invention forthe antibodies described in these methods.

The detectable label may be a fluorescent label, a chemiluminescentlabel, or a biolumincent label. Examples of fluorescent labels includefluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,allophycocyanin, o-phthaldehyde or fluorescamine. Examples ofchemiluminescent labels include luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester.Examples of biolumincent labels include luciferin, luciferase oraequorn.

The detectable labels may include one or more image enhancing agents.Image enhancing agents are useful for magnetic resonance imaging (MRI).Magnetic resonance imaging (MRI) agents are described, for example, inPykett, Scientific American, 246, 78(1982); Runge et al., Am. J.Radiol., 141, 1209(1983). Examples of compounds useful for MRI imageenhancement include complexes of paramagnetic ions, e.g., Gd(III),Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III),Cu(II), Ni(II), Ti(III), and V(IV) ions, or radicals, e.g., nitroxides,and these may be further attached to a substrate via a suitable linker.The MRI enhancing agent must be present in sufficient amounts to enabledetection by an external camera, using magnetic field strengths whichare reasonably attainable and compatible with patient safety andinstrumental design. The requirements for such agents are well known inthe art for those agents which have their effect upon water molecules inthe medium, and are disclosed, inter alia, in, e.g., Pykett, ScientificAmerican, 246:78 (1982); and Runge et al., Am. J. Radiol, 141:1209(1987).

The detectable label may comprise one or more radiopaque or contrastagents for X-ray or computed tomography. Radiopaque or contrast agentsmay be barium, diatrizoate, ethiodized oil, gallium citrate, iocarmicacid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, or thallouschloride. See U.S. Pat. Nos. 5,120,525, 5,128,119, 5,328,679.

The detectable label may comprise one or more ultrasound contrast agentssuch as, for example, a liposome (including gas filled liposome) ordextran.

In addition to the described detectable label, the PPC may comprise atherapeutic agent such as a radionuclide. Other applicable methods forlabeling the PPC of this invention are disclosed in U.S. Pat. Nos.5,061,641, 5,101,827.

Additional examples for using radiolabeled antibodies and engineeredantibodies for detection or therapy may be found in U.S. Pat. Nos.4,624,846, 5,482,698, 5,525,338, 5,609,846, 5,716,595, 5,728,369,5,736,119, 5,746,996, 5,772,981, 5,776,093, 5,776,094, 5,776,095,5,843,397, 5,851,527, 5,958,408, 5,965,131, 6,010,680, 6,077,499,6,096,289, 6,331,175, 6,361,774, 6,387,350, 6,399,068, and 6,458,933.The PPC of the invention may be substituted for any of the antibodiesmentioned in these patents.

The invention also provides for PPC where at least one ABS of the PPC isspecific for an epitope on a cancer associated antigen and at least oneABS is specific for an epitope on a hapten.

Another embodiment of the invention is directed to a PPC linked to aconjugate (See U.S. Pat. No. 4,824,659 for a description of an antibodyconjugate). The linkage may by a crosslinker. The conjugate may be aradionuclide or a cytotoxic agent, a drug, a chemotherapeutic agent, anda radionuclide.

In another embodiment of the invention, the PPC may have at least oneABS with specificity for an antigen on the surface of effector cells andat least one ABS specific for an antigen on a target cell or a virus.Examples of the first antigen may be an antigen of the surface ofT-cells, natural killer cells, granulocytes, monocytes, or macrophages.In this case, the binding of the PPC to these two antigens may result inthe killing or the mitotic arrest of the target cell. The followingarticles make reference to the utility of a polyvalent protein withthese characteristics: Takemura et al., Cancer Immunol. Immunother. 2002March; 51(1): 33-44; Kipriyanov et al., J. Immunol. 2002 Jul. 1; 169(1):137-44; Stockmeyer et al., J. Immunol 2000 Nov. 15; 165(10): 5954-61.

In another embodiment of the invention, the PPC may have at least oneABS with specificity for an antigen on the surface of a cells and atleast one ABS specific for an antigenic substance. Examples of the firstantigen may be an antigen of the surface of B-cells, monocytes,dendritic cells and macrophages. In this case, the binding of the PPC tothe cell surface antigen and the antigenic substance results in theinduction of an immune response to the antigenic substance.

In another embodiment, the invention is directed to a PPC wherein one ofthe polypeptides comprise an additional V-region. The V-region may belinked to the other V-regions by an additional linker. This additionalV-region may comprise an amino acid sequence of a toxin, a hapten or adetectable moiety. Many examples of toxins, haptens, and detectablemoiety are proteins and peptides with known amino acid sequences. Manyof these peptides are cited as examples throughout this specification.These amino acid sequences may be used in the V-regions described inthis paragraph.

Bispecific and Multispecific PPC

The invention provides for methods of using the PPC. In general, anymethod that require the use of an antibody or engineered antibody (see,e.g., Cao Y and Lam, L. Adv Drug Deliv Rev 2003 Feb. 10;55(2): 171-97)may be performed using a PPC with a similar binding affinity andspecificity. These methods includes any of the methods described in thisdisclosure and in the references, patents and patent applications citedtherein. Descriptions of specific embodiments are described below.

Bispecific and multispecific PPC are effective for the recruitment ofeffector functions and treatment of tumor cells. Multispecificity refersto the ability of a engineered antibody, like the PPCs of the invention,to have multiple ABS where each ABS binds a different epitope. Asdiscussed above, the fusion PPC of the invention may have at least 3 to10 or more ABS and each ABS may have specificity to a different epitope.Further each different epitope may be on the same or different antigen.Bispecific antibodies have found particular use in recruiting thepowerful effector functions of cytotoxic T cells or natural killer (NK)cells. Thus bispecific antibodies have been used to bridge the T cellcoreceptor (CD3) (Staerz et al., Nature 314: 628-631, 1985) or FcRIII(CD16) (De Palazzo et al., Cell Immunol. 142: 338-347, 1992) and thecell surface antigen of a target cell to mediate the killing of targetcells by cytotoxic T cells or NK cells. In mice, such anti-CD3 bisAbscan inhibit the growth of solid tumors (Titus et al., J. Immunol. 138:4018-4022, 1987, Garrido et al., Cancer Res. 50: 4227-4232, 1990) oreven eradicate lymphoma (Brissinck et al., J. Immunol 147: 4019-4026,1991); in humans, they have been used against malignant glioma(Brissinck et al., J. Immunol. 147: 4019-4026, 1991). Bispecificantibodies have also been used for ex vivo purging of leukaemia cellsfrom bone marrow (T. Kaneko et al., Blood 81: 1333-1341, 1993).Bispecific antibodies synthesized in vitro have also been used todeliver enzymes, antigens, toxins, radionuclides and cytotoxic drugs totumor cells (see Bonardi et al., Cancer Res. 53: 187-199 1992). Any ofthe above method, and any of the methods in the cited references in thisdisclosure, may be performed using the bispecific PPCs of the inventionas a substitute for the multispecific antibody (or functionalderivatives) specified in these method.

The multispecific PPC of the invention may be used for imaging oftumors. Bispecific anti-tumor marker, anti-hapten antibodies have beenused to image tumors (J. M. Le Doussal et al. Int. J. Cancer Supplement7: 58-62, 1992; P. Peltier et al. J. Nucl. Med. 34: 1267-1273 1993; C.Somasundaram et al. Cancer Immunol. Immunother. 36: 337-345, 1993; A.Bruynck et al. Br. J. Cancer 67: 436-440, 1993). The method comprisestwo steps. In the first step, a bispecific antibody is injected andlocalize to the tumor by binding to a tumor-associated antigen. In thesecond step, a radioactively labeled hapten is then injected whichpreferentially localizes to the tumor, by binding to the bispecificantibody, enabling imaging of the tumor. Multispecific PPC of theinvention with at least one ABS specific for tumor cells and one ABSspecific for the hapten could be used to in place of the bispecificantibody to achieve the same results.

As another example, the PPC of the invention may be used to delivercytotoxic drugs to tumor cells, using one binding site to deliver thedrug and the other to bind to the tumor, or using systems analogous tothat described for the delivery of doxorubicin to tumors by P. A. Trailet al. (Science 261: 212-215, 1993). These authors used an antibodydirected to the Lewis Y antigen, covalently linked to doxorubicin, whichwas internalized into lysosomes and endosomes. The linkage was cleavablein these environments leading to delivery of the drug to these cells.Bivalent PPCs may be particularly useful to increase the avidity of theantibody for the tumor cell. The specificity may be increased by using abispecific antibody directed against two (or more) differenttumor-associated antigen on the same tumor or two (or more) epitopes onthe same tumor-associated antigen.

The multispecific PPCs of the invention may be used to delivertherapeutic agents across the blood brain barrier. In this method, amultispecific PPC with one ABS directed against either FHA, an adhesinof the bacterium Bordetella pertussis or against the natural ligand forthe leucocyte adhesion molecule CR3 (E. I Tuomanen et al. Proc. Natl.Acad. Sci. USA 90: 7824-7828, 1993) and the other ABS may then bedirected against a target to provide the therapeutic function.

Multivalent PPCs may be particularly useful for imaging purposes forinstance when localizing tumors by binding to two different epitopes ofa tumor-associated antigen with a radiolabeled PPC. The presence of twoABS for one tumor-associated antigen would give an avidity componentwhich may increase the signal to noise ratio of the detection method.

The multispecific PPCs of the invention may be used in retargetting ofantibodies to a site or antigen for which they have no specificity undernormal circumstances. The PPC would possess two ABSs; one ABS isspecific for a target site, the other ABS is capable of binding toselected parts of an antibody molecule. In this manner, antibodies withno specificity for the antigen target are brought into proximity withthe antigen via the PPC. This principle is advantageous for re-targetingantibodies in the circulation to sites within the body such as tumorsand to block inappropriate immune responses exemplified by autoimmunedisease and would allow recruitment of effector functions.

In this way, multispecific PPCs could be used to recruit effectorfunctions through binding to whole antibody chains. One ABS of the PPCwould be directed against antigen for therapy and the second arm againstwhole antibodies for the recruitment of effector functions.

In a preferred embodiment of the invention, the ABS in a PPC may bespecific for an epitope of a tumor-associated antigen. Thetumor-associated antigen may be associated with, for example,carcinomas, melanomas, sarcomas, gliomas, leukemias or lymphomas. Atumor-associated antigen may have more than one epitope. For example, atumor-associated antigen may have at least 1, 2, 3, 4 epitopes. Othertarget antigens present in more than once cell type and useful in thisinvention are CD74, HLA-DR, Where the construct contain more than oneABS, the ABS may be specific for epitopes on the same tumor antigen ordifferent tumor antigens. Thus, a PPC with 3 or 4 ABSs may bind from 1to 3 or 4 of tumor antigens.

In a preferred embodiment, the ABS of a PPC has the same bindingspecificity as monoclonal antibody (Mab) Mu-9 and MAb 679. This can beachieved, for example, by using the sequence of the monoclonalantibodies to construct the V_(H) and V_(L) regions of the PPC.

In addition, the PPC of the invention may comprise one or more ABS whichbind an epitope on a hapten. The hapten may be ahistamine-succinyl-glycine (HSG) or indium-DTPA. Naturally, the ABS ofthe PPC may bind multiple epitopes of one hapten or different epitopesof different haptens. The three or more ABS of a PPC can bind anycombination of tumor-associated antigens or haptens without limitation.As an example, one ABS may bind CEA while another ABS may bind a non-CEAtumor-associated antigen. For example, where the number of ABS is equalto N, the number of ABS that binds tumor-associated antigen may rangefrom zero to N. The remainer of the ABS may all bind hapten.

As the above examples illustrate, the multispecific PPC of the inventionmay serve as a substitute for multi specific engineered antibodies inany method. These methods includes any of the methods described in thisdisclosure and in the references, patents and patent applications citedtherein. More specific examples of the use of PPC are discussed below.

Methods for Treating, Diagnosing, and Detecting Disorders

The invention also provides for methods for treating, diagnosing, anddetecting a symptom of a neoplastic disorder by administering any of thePPC of this disclosure with an ABS directed to a cancer associatedantigen. The PPC may be administered with one or more therapeuticagents, diagnostic agents, or detecting agents and one or morecytokines. The therapeutic agent may be a chemotherapeutic agent or acombination of chemotherapy agents. The administration of thetherapeutic agent or cytokine may be before, during or after theadministration of the PPC.

When more than one therapeutic agents are used, the therapeutic agentsmay be the same or different, and may be, for example, therapeuticradionuclides, drugs, hormones, hormone antagonists, receptorantagonists, enzymes or proenzymes activated by another agent,autocrines or cytokines. Toxins also can be used in the methods of thepresent invention. Other therapeutic agents useful in the presentinvention include anti-DNA, anti-RNA, radiolabeled oligonucleotides,such as anti-sense oligonucleotides, anti-protein and anti-chromatincytotoxic or antimicrobial agents. Other therapeutic agents are known tothose skilled in the art, and the use of such other therapeutic agentsin accordance with the present invention is specifically contemplated.

In a preferred embodiment, the therapeutic agents comprise differentisotopes, which are effective over different distances as a result oftheir individual energy emissions, are used as first and secondtherapeutic agents. This process achieves more effective treatment oftumors, and is useful in patients presenting with multiple tumors ofdiffering sizes, as in normal clinical circumstances.

Few of the available isotopes are useful for treating the very smallesttumor deposits and single cells, and a drug or toxin may be a moreuseful therapeutic agent in these situations. Accordingly, in preferredembodiments of the present invention, isotopes are used in combinationwith non-isotopic species such as drugs, toxins, and neutron captureagents. Many drugs and toxins are known which have cytotoxic effects oncells, and can be used in connection with the present invention. Theyare to be found in compendia of drugs and toxins, such as the MerckIndex, Goodman and Gilman, and the like, and in the references citedabove.

Drugs that interfere with intracellular protein synthesis can also beused in the methods of the present invention; such drugs are known tothose skilled in the art and include puromycin, cycloheximide, andribonuclease.

The therapeutic agents may be linked to the PPC. Methods of makinglinked proteins in which one recombinant protein comprises a cytotoxicagent, therapeutic agent or chemotherapeutic agent also are known tothose of skill in the art. These methods can be applied to the PPC ofthe invention. For example, antibody-Pseudomonasexotoxin A PPCs havebeen described by Chaudhary et al., Nature 339: 394 (1989), Brinkmann etal., Proc. Nat'l Acad. Sci. USA 88: 8616 (1991), Batra et al., Proc.Nat'l Acad. Sci. USA 89: 5867 (1992), Friedman et al., J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al.,J. Biol. Chem. 271: 10560 (1996), Kuan et al., Biochemistry 35: 2872(1996), and Schmidt et al., Int. J. Can. 65: 538 (1996). Antibody-toxinPPCs containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7: 553 (1993), Nicholls et al., J. Biol. Chem.268: 5302 (1993), Thompson et al., J. Biol. Chem. 270: 28037 (1995), andVallera et al., Blood 88: 2342 (1996). Deonarain et al., Tumor Targeting1: 177 (1995), have described an antibody-toxin PPC having an RNasemoiety, while Linardou et al., Cell Biophys. 24-25: 243 (1994), producedan antibody-toxin PPC comprising a DNase I component. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91: 8945 (1994),reported an antibody-toxin PPC comprising Staphylococcal enterotoxin-A.These methods are also applicable for making the PPCs comprising a toxinof the invention. Other suitable cytotoxic agents are listed in thedefinitions section of this disclosure.

It is to be understood that any combination of the above describedtherapeutic agents may be used. For example, a PPC may be conjugated totwo or more radioisotopes, or drugs. When a mixture of therapeuticagents is used, a plurality of therapeutic agents are delivered to thetumor sites, thereby enhancing the benefits of the method. The use ofmixtures of nuclides has the further advantage that a greater percentageof the injected biotinylated chelates delivers a toxic payload to thetumor target.

The present invention also contemplates dyes used, for example, inphotodynamic therapy, and used in conjunction with appropriatenon-ionizing radiation. The use of light and porphyrins in methods ofthe present invention is also contemplated and their use in cancertherapy has been reviewed (van den Bergh, Chemistry in Britain, 22:430-437 (1986)).

The invention also provides for methods of reducing a symptom of aneoplastic disorder in a subject. The subject can be any animalincluding horses, mice, rats, pigs, bovines, chickens etc. In apreferred embodiment, the animal is a human. In the method, a PPC isadministered to a patient displaying a symptom of the neoplasticdisorder to reduce the symptom. The neoplastic disorder may be acarcinomas, sarcomas, gliomas, lymhomas, leukemias, melanomas or thelike. In a preferred embodiment, the neoplastic disorder is a B-cellmalignancy such as indolent forms of B-cell lymphomas, aggressive formsof B-cell lymphomas (including non-Hodgkin's lymphoma), chroniclymphatic leukemias, or acute lymphatic leukemias.

Another embodiment of the invention is directed to a method for treatingB cell malignancies. The method involves administering to a subjecthaving a B cell malignancy one or more dosages of a therapeuticcomposition which contains a pharmaceutically acceptable carrier and atleast one PPC of the invention. The B-cell malignancies may be anyB-cell malignancy including, at least, carcinomas, sarcomas, gliomas,lymphomas, leukemias, and melanomas. In the method, the PPC may beparenterally administered in a dosage of 20 to 1500 milligrams proteinper dose. In a preferred embodiment, the PPC may be administered in adosage of 20 to 500 milligrams protein per dose. In a most preferredembodiment, the PPC may be parenterally administered in a dosage of 20to 100 milligrams protein per dose. Any of these dosages may berepeatedly administered to achieve an even higher dosage. As discussedabove, the PPC of the invention, including the PPC in the methods of theinvention, may be radiolabeled. In administering a PPC that isradiolabeled, the dosage of the radiolabel may be between 15 to 40 mCi.In a preferred embodiment, the dosage is between 10 and 30 mCi. In amore preferred embodiment, the dosage may be between 20 and 30 mCi. Inanother more preferred embodiment, the dosage may be between 10 and 20mCi.

In another embodiment, where a method of the calls for theadministration of PPC, the PPC may be administered before, after orconcurrently with a chemotherapeutic agent, cytokine, or colonystimulating factor. Specific examples of chemotherapeutic agents andcytokines are enumerated in another section of the specification.

Any of the methods of the invention, including methods for treatingautoimmune disorders and neoplastic disorders may be used to treatdisorders such as cardiovascular diseases and inflammation. Thesedisorders include clots, emboli, myocardial infarction, ischemic heartdisease, and atherosclerotic plaques. PPCs that are suitable fortreating these disorders include those PPCs with an ABS specifc for CD74(e.g., hLL1), NCA (or -CD66) and NCA90. This would include ABS with thesame specificity as hMN3. The diagnostic imaging methods of theinvention are particularly adaptable for using the above stated PPC. Inparticular, the detection methods, diagnostic methods, and the cellablation methods may be applied to cardiovascular disorders. Forexample, the detection may be used to detect damaged heart and vasculartissue. The cell ablation methods may be used for targeting diseasedheart tissue. Inflammation can be detected or treated withanti-granulocyte (e.g., anti-CD66, anti-CD33, anti-CD45),anti-lymphocyte (anti-B- or anti-T-cell antibodies), and/oranti-monocyte antibodies (e.g., anti-Ia or anti-CD74 antibody).

In another embodiment of the invention, the treatment methods of theinvention can be used in combination with other compounds or techniquesfor preventing, mitigating or reversing the side effects of cytotoxicagents. Examples of such combinations include, e.g., administration ofIL-1 together with a second antibody for rapid clearance, as described.e.g., U.S. Pat. No. 4,624,846, from 3 to 72 hours after administrationof a targeted primary PPC antibody fragment conjugate (with aradioisotope, drug or toxin as the cytotoxic component of theimmunoconjugate) or of a non-conjugated drug or toxin, to enhanceclearance of the conjugate, drug or toxin from the circulation and tomitigate or reverse myeloid and other hematopoietic toxicity caused bythe therapeutic agent. This method is also applicable to the PPC of theinvention.

In another aspect, cancer therapy often involves a combination of morethan one tumoricidal agent, e.g., a drug and a radioisotope, or aradioisotope and a Boron-10 agent for neutron-activated therapy, or adrug and a biological response modifier, or a PPC conjugate and abiological response modifier. The cytokine can be integrated into such atherapeutic regimen to maximize the efficacy of each component thereof.

Similarly, certain antileukemic and antilymphoma antibodies conjugatedwith radioisotopes that are β or α emitters can induce myeloid and otherhematopoietic side effects when these agents are not solely directed tothe tumor cells, particularly when the tumor cells are in thecirculation and in the blood-forming organs. Concomitant and/orsubsequent administration of the hematopoietic cytokine (growth factor,such as colony stimulating factors (e.g., G-CSF and GM-CSF) is preferredto reduce or ameliorate the hematopoietic side effects, while augmentingthe anticancer effects.

In addition to preventing, mitigating or reversing the myelosuppressiveor other hematopoietic side effects of the therapy, cytokines such as,e.g., IL-1, can have anticancer effects (Nakamura et al., Gann77:1734-1739, 1986; Nakata et al., Cancer Res. 48:584-588, 1988), aswell as IL-12, and therefore are capable of enhancing the therapeuticeffect of the targeted agents when used in combination with these othertherapeutic modalities. Thus, another aspect of the present invention isto maximize the antiproliferative activity of the cytokine byconjugating it to the targeting PPC to form a heteroconjugate. Since thecytokines are polypeptides, conjugation to the PPC can be performedusing any of the conventional methods for linking polypeptides toantibodies. These include, e.g., use of the heterobifunctional reagentN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), according topublished procedures, e.g., that of Carlsson et al., Biochem. J.173:723-737, 1978, use of glutaraldehyde, carbodiimide or likecondensing and/or linking reagents.

It is preferable to achieve a high ratio of the cytokine to the PPCwithout affecting the latter's immunoreactivity and targetingproperties. Thus, it may be advantageous to use a carrier for thecytokine and to link a plurality of cytokine molecules to the carrier,which is then linked to the PPC. A particularly effective method forachieving this result is to use the method of Shih et al., PCT/US WO87/005031, wherein an addend is conjugated to a polymer such as anaminodextran, which is then site-specifically linked to the oxidizedcarbohydrate portion of a PPC. Depending upon the cytokine and PPC used,20 to more than 100 cytokine molecules per PPC can be attached withoutaffecting the PPC appreciably, and in some circumstances 100 to 1,000molecules of cytokine per PPC molecule can be achieved.

Use of IL-1 or G-CSF as the cytokine is preferable if a cytokine withantitumor activity is desired to potentiate the targeting PPC's effects,especially if the latter is conjugated with a toxic radioisotope ordrug. If the targeting PPC circulates and deposits in other normalorgans, such as the bone marrow, then the presence of the cytokine isimportant to prevent, mitigate or reverse the hematologic side effectsthat would normally result. Since some of the cytokines have lymphoideffector cell functions for tumor cell killing (e.g., IL-2), theheteroconjugate of this invention provides a multimodal therapy to thetarget, whether it be a cancer, an infection, or another lesion that isunresponsive to more traditional measures.

An appropriate dose of the cytokine can be administered prior to,simultaneously with or subsequent to the administration of thetherapeutic agent. The object will be to maximize the cytotoxic activityof the agent on the pathological lesion, such as cancer cells orinfectious organisms, while minimizing toxicity to the myeloid and otherhematopoietic cells. Careful monitoring of the WBC and other bloodelements, including but not limited to erythrocyte (red blood cell/RBC)count, thrombocyte (platelet) count, and including a differential WBCanalysis to monitor the myloid/lymphoid series, as well as the bonemarrow hematological picture during the course of therapy, withparticular attention to possible depletion of myeloid lymphoid forms,but also the status of immature erythrocytes, myelocytes, lymphocytesand thrombocytes, will permit optimization of the cytokine treatment.Depending upon which hematologic element is adversely affected, thechoice of cytokine and administration schedule can be individualized ineach circumstance, including the combination of cytokines, such as IL-1and IL-3; IL-1 and IL-2; IL-1 and GM-CSF; IL-1, erythropoietin, andplatelet growth factor and the like.

Correlation of the choice of cytokine, singly or in combinations, anddoses thereof, to hematotoxicity is important, since each cytokinegenerally has its effect mostly on particular hematopoietic elements.The following guidelines may be used for choosing cytokines in themethods of the invention. For example, if a cytotoxic agent has bothsevere myeloid and thrombocytic toxicity, the combination of IL-1 andIL-3 in a 1:1 or 2:1 (or higher) ratio will be advantageous. Thus,reduction in the WBC count to a level below about 2,000 and platelets toa level below about 20,000 can be reversed by administration of fromabout 1 ug to about 500 ug, preferably 5-100 ug, more preferably about10 ug of rIL-1 in a single dose, together with or followed byadministration of from about 1 ug to about 200 ug, preferably 5-50 ug,more preferably about 5 ug of IL-3. The applications can be repeated,with the reversal of the myeloid and platelet depressions occurringwithin about 5-20 days, usually about 7 days. The ordinary skilledclinician will appreciate that variations in the timing and dosage ofcytokine administration and cytokine combinations and dosages are afunction of the cytokine used, the nature of the bone marrow and/orother hematopoietic element depressed, and the nature of the patient(e.g., prior toxicity affecting bone marrow status) and the cytotoxicagent and protocol.

Examples of autoimmune diseases that could be treated by the methods ofthe invention include acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcalnephritis,erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pemiciousanemia, rapidly progressive glomerulonephritis, psoriasis, andfibrosing alveolitis.

The method of treating autoimmune disease may comprise an additionalstep of administering a secondary antibody or PPC with an ABS specificfor an epitope on T-cells, plasma cells, or macrophages or inflammatorycytokines. This additional step may be performed before, during or afterthe administration the PPC.

Another major application of the methods and PPCs of the invention is todepress host immunity in certain autoimmune diseases such as, forexample, systemic lupus erythematosis, and in patients receiving organtransplants. In these applications, the PPC is associated with cytotoxicdrugs. These cytotoxic drugs are similar to those often used in cancerchemotherapy, with the attendant myeloid and other hematopoietic sideeffects. In addition to these drugs, specific PPCs targeted againstthese lymphoid cells (particularly T-cell), (e.g., a PPC with an ABSderived from the anti-Tac monoclonal antibody of Uchiyama et al., J.Immunol. 126:1393 and 1398 (1981), which specifically binds to the humanIL-2 receptor of activated T-cells) can be conjugated to cytotoxicagents, such as drugs, toxins or radioisotopes, to effect a relativelyselect killing of these cells involved in organ rejection. For example,a T-cell specific PPC can be conjugated with α, β or γ emittingradioisotope, and this can be administered to the patient prior toundertaking organ transplantation and, if needed, also thereafter.

In order to effect a high T-cell killing dose without the concomitantlimiting side effects to the hematopoietic system, this treatment can becombined with the use of cytokines, according to the present invention.This method is preferred for the long-term survival of many organtransplants, such as the kidney, heart, liver, etc., where a criticalperiod of organ rejection needs to be overcome.

The dosage level of the cytokine will be a function of the extent ofcompromise of the hematopoietic cells, correlated generally with thewhite blood cell (WBC) level in the patient. Periodic monitoring of theWBC and other blood cell counts and adjustment of the rate and frequencyof infusion or the dosage of the cytokine administered to achieve arelatively constant level of WBC's will ensure that the patient does notsustain undue marrow toxicity from the therapy. Experience will permitanticipation of WBC lowering and in fusion of the cytokine at a time andin an amount sufficient to substantially prevent WBC-depression.Importantly, this also insures that excessive side effects due to thecytokine itself are not produced, but only such side effects as arenecessary to prevent compromise of the patient's myeloid and otherhematopoietic cell systems.

Correlation of cytokine dosage to WBC count suggests that, in general,reduction of WBC count from a normal range of 8-12,000/mm³ to a level ofabout 2,000 can be reversed by administration of from about 1 ug toabout 500 ug, preferably 5-100 ug, more preferably about 10 ug ofrecombinant human IL-1 in a single dose, the reversal of WBC countdepression occurring within about 2-12 days, usually about 4 days. Theclinician will appreciate that variations in the timing and dosage ofcytokine administration as a function of the type of cytokine used, theextent and rate of compromise of the bone marrow and/or other componentsof the myeloid and/or other hematopoietic elements and the individualcharacteristics of the patient and the therapy protocol will be possibleand often desirable. These can easily be made by the clinician usingconventional monitoring techniques and dosimetric principles.

The methods of the invention, including methods for treating autoimmunedisorders and neoplastic disorders may be used to treat disorders suchas cardiovascular diseases and inflammation. These disorders includemyocardial infarction, ischemic heart disease, and atheroscleroticplaques. PPCs that are suitable for treating these disorders includethose PPCs with an ABS specific for CD74 (e.g., hLL1), NCA (or -CD66)and NCA90. This would include ABS with the same specificity as hMN3. Thediagnostic imaging methods of the invention are particularly adaptablefor using the above stated PPCs. In particular, the detection methods,diagnostic methods, and the cell ablation methods may be applied tocardiovascular disorders. For example, the detection may be used todetect damaged heart and vascular tissue. The cell ablation methods maybe used for targeting diseased heart tissue. Inflammation can bedetected or treated with anti-granulocyte (e.g., anti-CD66, anti-CD33,anti-CD45), anti-lymphocyte (anti-B- or anti-T-cell antibodies), and/oranti-monocyte antibodies (e.g., anti-Ia or anti-CD74 antibody).

Any of the methods of the invention, including methods for treatingautoimmune disorders and neoplastic disorders may be used to treatdisorders such as neurological diseases such as Alzheimer's disease. PPCthat are suitable for treating these disorders include those PPC with anABS specific for the amyloid plaques of Alzheimer patients. Thediagnostic imaging methods of the invention are particularly adaptablefor using the above stated PPC. In particular, the detection methods,diagnostic methods, and the cell ablation methods may be applied toneurological disorders. For example, the detection may be used to detectdamaged brain tissue or brain tissue with amyloid. The cell ablationmethods may be used for targeting amyloid. Inflammation can be detectedor treated with anti-amyloid PPCs.

This invention also provides for methods of detecting and diagnosing adiseased tissue or a disease. For example, any of the methods oftreatment presented may be performed with a PPC that has a detectablelabel, such as, for example, a radiolabel. The PPC can be detected afteradministration to the patient. Thus, any of the treatment methods can beused as detecting methods by the additional step of detecting the PPCafter administration to the patient. Furthermore, by using a PPC with aspecificity to a known pathogen, diseased cell, tumor associatedantigen, disease associated antigen (e.g., amyloid) and the like, thepresence of a disease may be diagnosed.

Methods of Administration

The preferred route for administration of the invention is parentalinjection. In parenteral administration, the compositions of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives. Other methodsof administration, such as oral administration are also contemplated.

The PPC of the present invention may be administered in solution. The pHof the solution should be in the range of pH 5 to 9.5, preferably pH 6.5to 7.5. The PPC thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate, tris(hydroxymethyl) aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The solution ofthe immunoglobulin may also contain a salt, such as sodium chloride orpotassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as glycerol, albumin, a globulin, adetergent, a gelatin, a protamine or a salt of protamine may also beincluded and may be added to a solution containing the PPC or to thecomposition from which the solution is prepared. Systemic administrationof the PPC is typically made every two to three days or once a week if ahumanized form of the antibody is used as a template for the PPC.Alternatively, daily administration is useful. Usually administration isby either intramuscular injection or intravascular infusion.

Administration may also be intranasal or by other nonparenteral routes.The PPC may also be administered via microspheres, liposomes or othermicroparticulate delivery systems placed in certain tissues includingblood.

The PPC may be administered by aerosol to achieve localized delivery tothe lungs. This is accomplished by preparing an aqueous aerosol,liposomal preparation or solid particles containing or derivativesthereof. A nonaqueous (e.g., fluorocarbon propellent) suspension couldbe used. Sonic nebulizers preferably are used in preparing aerosols.Sonic nebulizers minimize exposing the PPC to shear, which can result indegradation of the PPC.

In general, the dosage of administered PPC will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Preferably, a saturating dose ofPPC is administered to a patient.

Typically, it is desirable to provide the recipient with a dosage thatis in the range of from about 50 to 500 milligrams of PPC, although alower or higher dosage also may be administered as circumstancesdictate. Examples of dosages include 20 to 1500 milligrams protein perdose, 20 to 500 milligrams protein per dose, 20 to 100 milligramsprotein per dose, 20 to 100 milligrams protein per dose, 20 to 1500milligrams protein per dose. In the embodiments where the PPC of PPCcomprise a radio nuclide, the dosage may be measured by millicurries. Inthat case, the dosage may be between 15 and 40 mCi, 10 and 30 mCi, 20and 30 mCi, or 10 and 20 mCi.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known to those inthe art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co.,New York, 1980 and 2001 editions).

For purposes of therapy, one or more PPCs and a pharmaceuticallyacceptable carrier are administered to a patient in a therapeuticallyeffective amount. A combination of one or more PPCs and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. In the present context, an agent is physiologicallysignificant if its presence results in the inhibition of the growth oftarget cells.

PPC linked to radionuclide are particularly effective for microbialtherapy. After it has been determined that PPC are localized atinfectious sites in a subject, higher doses of the labeled PPC,generally from 20 mCi to 150 mCi per dose for I-131, 5 mCi to 30 mCi perdose for Y-90, or 5 mCi to 20 mCi Re-186, each based on a 70 kg patientweight, are injected. Injection may be intravenous, intraarterial,intralymphatic, intrathecal, or intracavitary (i.e., parenterally), andmay be repeated. It may be advantageous for some therapies to administermultiple, divided doses of PPC or PPC composite, thus providing highermicrobial toxic doses without usually effecting a proportional increasein radiation of normal tissues.

A variety of radionuclides are useful for therapy, and they may beincorporated into the specific PPC by the labeling techniques discussedabove, as well as other conventional techniques well known to the art.Preferred therapeutically effective radionuclides are actinium-225,astatine-211, bismuth-212, yttrium-90, rhenium-186, rhenium-188,copper-67, phosphorus-32, lutetium-177, iodine-131, and iodine-125,although other radionuclides as well as photosensitizing agents are alsosuitable.

As discussed above, the PPC may be labeled and the use of a labeled PPCin the methods of the invention is also contemplated. The dosage of theradiolabel may be in the range of between 10 and 60 mCi per dose foryttrium-90. Preferably, between 10 and 50 mCi per dose. Most preferably,between 15 and 40 mCi, or between 20 to 30 mCi per dose or between 10 to30 mCi per dose of yttrium-90.

In a preferred embodiment of the invention, the PPC to be administeredto a patient suffering from a neoplastic disorder is an PPC comprisingat least one ABS specific for an epitope from the appropriatetumor-associated antigen. That is, the PPC can bind one of thesetumor-associated antigens at more than one site (epitope). Bispecificand polyspecific immunoglobulin derivative have may uses which areenumerated in WO02082041A2.

In practicing the methods of the invention the PPC may further compriseany of the additional components described above which includes, atleast, toxins, radionuclide, chemotherapeutic agents or antimicrobialagents. In some embodiments of the invention, a chemotherapeutic agentmay be physically linked to the PPC. In other embodiments, thechemotherapeutic agent may be unlinked. Unlinked chemotherapeutic agentsmay be administered before, during, or after the administration of thePPC.

In another embodiment of the invention, the PPC may be administeredbefore, during, or after administration of a cytokine moiety. Otheragents that can be advantageously administered before, during of afterthe administration of PPC, for any of the methods of the invention,include at least, granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF),erythropoietin, thrombopoietin, and the like. Other useful agentsinclude a hematopoietic growth factors conjugated to a bispecificantibody.

The PPC of the invention may be substituted for immunoglobulin used forcancer therapy. It is well known that radioisotopes, drugs, and toxinscan be conjugated to antibodies or antibody fragments which specificallybind to markers which are produced by or associated with cancer cells,and that such antibody conjugates can be used to target theradioisotopes, drugs or toxins to tumor sites to enhance theirtherapeutic efficacy and minimize side effects. Examples of these agentsand methods are reviewed in Wawrzynczak and Thorpe (in Introduction tothe Cellular and Molecular Biology of Cancer, L. M. Franks and N. M.Teich, eds, Chapter 18, pp. 378410, Oxford University Press. Oxford,1986), in Immunoconjugates. Antibody Conjugates in Radioimaging andTherapy of Cancer (C. W. Vogel, ed., 3-300, Oxford University Press,N.Y., 1987), in Dillman, R. O. (CRC Critical Reviews inOncology/Hematology 1:357, CRC Press, Inc., 1984), in Pastan et al.(Cell 47:641, 1986). in Vitetta et al. (Science 238:1098-1104, 1987) andin Brady et al. (Int. J. Rad. Oncol. Biol. Phys. 13:1535-1544, 1987).Other examples of the use of immunoconjugates for cancer and other formsof therapy have been disclosed, inter alia, in U.S. Pat. Nos. 4,331,647,4,348,376,4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,5614,624,846, 4,818,709, 4,046,722, 4,671,958, 4,046,784, 5,332,567,5,443,953, 5,541,297, 5,601,825, 5,635,603, 5,637,288, 5,677,427,5,686,578, 5,698,178, 5,789,554, 5,922,302, 6,187,287, and 6,319,500.These methods are also applicable to the methods of the invention by thesubstitution of the immunoconjugated engineered antibodies andantibodies of the previous methods with the PPC of this invention.

The PPC of the invention, for use in any of the methods of theinvention, may be associated or administered with antimicrobial agents.

The PPC of the invention, for use in any of the methods of theinvention, may be associated or administered with cytokines and immunemodulators (defined elsewhere in this disclosure). These cytokines andimmune modulators, includes, at least, interferonss alpha, beta andgamma, and colony stimulating factors.

The invention also provides for methods for stimulating the immuneresponse in a patient using the PPC of the invention. In one embodiment,the PPC of the invention may comprise an ABS of an anti-idiotypeantibody. The PPC may mimic an epitope of a tumor-associated antigen toenhance the body's immune response.

The PPC of the invention may be used for many immunological procedurescurrently employing antibodies. These procedures include the use ofanti-idiotypic antibodies and epitope conjugated antibodies to boost theimmune system. See, U.S. Pat. Nos. 5,798,100 6,090,381, 6,132,718.Anti-idiotypic antibodies are also employed as vaccines against cancersand infectious diseases U.S. Pat. Nos. 6,440,416 and 6,472,511. Furtherpolyspecific PPC may bind multidrug transporter proteins and overcomemultidrug resistant phenotype in cells and pathogens. The antibodies inthese methods may be replaced by the PPC of this invention.

The invention also provides for a method for treating a symtom of anautoimmune disorder. In the method, an PPC of the invention isadministered to a patient with an autoimmune disorder. The PPC may beadmixed with a pharmaceutically acceptable carrier beforeadministration. The PPC of this method should contain at least one ABSwith binding specificity to a B-cell antigen epitope. The B cell antigenmay be CD22 and the epitope may be epitope A, epitope B, epitope C,epitope D and epitope E of CD22. The B cell-associated antigen may alsobe another cell antigen such as CD 19, CD20, HLA-DR and CD74.

The ABS may contain a sequence of subhuman primate, murine monoclonalantibody, chimeric antibody, humanized antibody, or human origin. Forexample, the ABS may be of humanized LL2 (anti-CD22) or A20 (anti-CD20)monoclonal antibody origin.

The administration may be parenteral with dosages from 20 to 2000 mg perdose. Administration may be repeated until a degree of reduction insymptom is achieved.

The patients who may be treated by the methods of the invention includeany animal including humans. Preferably, the animal is a mammal such ashumans, primates, equines, canines and felines.

The method and PPCs of the invention may be used for the treatment ofdiseases that are resistant or refractory towards systemic chemotherapy.These include various viral, fungal, bacterial and protozoan infections,as well as particular parasitic infections. Other viral infectionsinclude those caused by influenza virus, herpes virus, e.g.,Epstein-Barr virus and cytomegalovirus, rabies virus (Rhabdoviridae) andpapovavirus, all of which are difficult to treat with systemicantibiotic/cytotoxic agents. Use of PPC conjugates, provides asignificantly higher therapeutic index for antiviral drugs and toxins,thus enhancing their efficacy and reducing systemic side effects.Targeted radioimmunotherapy with conjugates of PPC with therapeuticradioisotopes (including boron addends activatable with thermalneutrons) offers a new approach to antiviral therapy

Protozoans that may be treated by the methods and PPCs include, e.g.,Plasmodia (especially P. falciparum, the malaria parasite), Toxoplasmagondii (the toxoplasmosis infectious agent), Leishmaniae (infectiousagent in leishmaniasis), and Escherichia histolytica. Detection andtreatment of malaria in its various stages is significantly enhancedusing the PPC of the invention. As noted above, MAbs that bind tosporozoite antigens are known. However, since sporozoite antigens arenot shared by blood stage parasites, the use of MAbs against sporozoiteantigens for targeting is limited to a relatively short period of timein which the sporozoites are free in the circulation, prior to and justafter injection of and development in the host's hepatocytes. Thus, itis preferable to use a mixture of PPCs. Alternatively, a PPC with ABSsthat target more than one parasite stage of a protozoan (such as P.falciparum) is also useful. The MAbs are conjugated to a suitableradionuclide for imaging (e.g., Tc-99m) or for therapy (e.g.,astatine-211; rhenium-186), or with an antimalarial drug (e.g.,pyrimethamine) for more selective therapy.

Toxoplasmosis is also resistant to systemic chemotherapy. It is notclear whether MAbs that bind specifically to T. gondii, or natural, hostantibodies, can play a role in the immune response to toxoplasmosis but,as in the case of malarial parasites, appropriately targeted PPC areeffective vehicles for the delivery of therapeutic agents.

Schistosomiasis, a widely prevalent helminth infection, is initiated byfree-swimming cercariae that are carried by some freshwater snails. Asin the case of malaria, there are different stages of cercariae involvedin the infectious process. PPCs that bind to a plurality of stages ofcercariae, optionally to a plurality of epitopes on one or more thereof,and preferably in the form of a polyspecific composite, can beconjugated to an imaging or therapy agent for effective targeting andenhanced therapeutic efficacy.

PPCs that bind to one or more forms of Trypanosoma cruzi, the causativeagent of Chagas' disease, can be made and used for detection andtreatment of this microbial infection. PPCs which reacts with acell-surface glycoprotein, as well as PPCs reactive with other surfaceantigens on differentiation stages of the trypanosome, are suitable fordirecting imaging and therapeutic agents to sites of parasiticinfiltration in the body.

Another very difficult infectious organism to treat by available drugsis the leprosy bacillus (Mycobacterium leprae). PPCs that specificallybind to a plurality of epitopes on the surface of M. leprae can be madeand can be used, alone or in combination, to target imaging agentsand/or antibiotic/cytotoxic agents to the bacillus.

Helminthic parasitic infections, e.g., Strongyloidosis and Trichinosis,themselves relatively refractory towards chemotherapeutic agents, aresuitable candidates for PPC-targeted diagnosis and therapy according tothe invention, using PPCs that bind specifically to one or, preferably,to a plurality of epitopes on the parasites.

Antibodies are available or can easily be raised that specifically bindto most of the microbes and parasites responsible for the majority ofinfections in humans. Many of these have been used previously for invitro diagnostic purposes and the present invention shows their utilityas components of antibody conjugates to target diagnostic andtherapeutic agents to sites of infection. Microbial pathogens andinvertebrate parasites of humans and mammals are organisms with complexlife cycles having a diversity of antigens expressed at various stagesthereof. Therefore, targeted treatment can best be effected when PPCconjugates which recognize antigen determinants on the different formsare made and used in combination, either as mixtures or as polyspecificconjugates, linked to the appropriate therapeutic modality. Theproduction of PPC is not difficult because the antibody may be purifiedand its sequence determined. The same principle applies to using thereagents comprising PPCs for detecting sites of infection by attachmentof imaging agents, e.g., radionuclides and/or MRI enhancing agents.

The invention also provides a method of intraoperatively identifyingdiseased tissues by administering an effective amount of a PPC; and atargetable construct where the PPC comprises at least one antigenbinding site that specifically binds a targeted tissue and at least oneother antigen binding site that specifically binds the targetableconstruct; and wherein said at least one antigen binding site is capableof binding to a complementary binding moiety on the target cells,tissues or pathogen or on a molecule produced by or associatedtherewith.

The invention also provides a method for the endoscopic identificationof diseased tissues, in a subject, by administering an effective amountof a PPC and administering a targetable construct. The PPC comprises atleast one antigen binding site that specifically binds a targeted tissueand at least one antigen binding site that specifically binds thetargetable construct; and wherein said at least one antigen binding siteshows specific binding to a complementary binding moiety on the targetcells, tissues or pathogen or on a molecule produced by or associatedtherewith.

An alternative method of detection suitable for use in the presentinvention is wireless capsule endoscopy, using an ingested capsulecamera/detector of the type that is commercially available from, forexample, Given Imaging (Norcross Ga.).

The invention also provides a method for the endoscopic identificationof diseased tissues, in a subject, by administering an effective amountof a PPC; and administering a targetable construct. In this embodiment,the PPC comprises at least one antigen binding site that specificallybinds a targeted tissue and at least one antigen binding site thatspecifically binds the targetable construct; and wherein said at leastone antigen binding site shows specific binding to a complementarybinding moiety on the target cells, tissues or pathogen or on a moleculeproduced by or associated therewith.

The invention also provides a method for the intravascularidentification of diseased tissues, in a subject by administering aneffective amount of a PPC and a targetable construct. The PPC compriseat least one antigen binding site that specifically binds a targetedtissue and at least one antigen binding site that specifically binds atargetable construct. The at least one antigen binding site is capableof binding to a complementary binding moiety on the target cells,tissues or pathogen or on a molecule produced by or associated with thecell, tissues or pathogen. The target tissue may be a tissue from normalthyroid, liver, heart, ovary, thymus, parathyroid, endometrium, bonemarrow, or spleen.

The invention also provides for a kit for practicing the methods of theinvention. The kit may include a targetable construct. The targetableconstruct may be labeled by any of the labels described as suitable fortargetable constructs above. Further, the targetable construct may beunlabeled but the kit may comprise labeling reagents to label thetargetable construct. The labeling reagents, if included, may containthe label and a crosslinker. The kit may also contain an PPC of theinvention comprising at least one ABS specific for the targetableconstruct and at least one ABS specific for a targetable tissue. The kitmay optionally contain a clearing composition for clearing non-localizedPPC.

Nucleic Acid Encoding PPC

Another embodiment of the invention is directed to a nucleic acidmolecule with at least one open reading frame (ORF) that encodes atleast one polypeptide of any of the PPC of the invention. The openreading frame of the nucleic acids of the invention may be linked, in anoperational manner, to one or more nucleic acid elements that promotethe expression of the open reading frame. These elements are known tothose of skill in the art and include, at least, promoters, enhancers,proximal stimulatory elements and the like. In a preferred embodiment, anucleic acid molecule may comprise two open reading frame that togetherexpress both chains of a PPC (see, e.g., FIG. 1A).

The nucleic acids of the invention may be present in many forms such as,for example, an expression cassette or an episome (plasmids, cosmids,and the like). Thus, an expression cassette or an episome, such as aplasmid or cosmid) is also envisioned as an embodiment of the invention.Another embodiment of the invention is directed to a host cellcomprising a nucleic acid of the invention. The host cell may be an E.Coli, a yeast, a plant cell or a mammalian cell. Mammalian cells may be,for example, a human cell or a mouse cell.

The nucleic acids of the invention may be expressed. Where the nucleicacid is an RNA, the expression may involve a first step of reversetranscribing the RNA into DNA. The DNA sequence may then be operablylinked to regulatory sequences controlling transcriptional expression inan expression vector and then, introduced into either a prokaryotic oreukaryotic host cell. In addition to transcriptional regulatorysequences, such as promoters and enhancers, expression vectors includetranslational regulatory sequences and a marker gene which is suitablefor selection of cells that carry the expression vector.

Suitable promoters for expression in a prokaryotic host can berepressible, constitutive, or inducible. Suitable promoters arewell-known to those of skill in the art and include promoters capable ofrecognizing the T4, T3, Sp6 and T7 polymerases, the PR and PL promotersof bacteriophage lambda, the trp, recA, heat shock, and lacZ promotersof E. coli, the α-amylase and the sigma-specific promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofthe β-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters arereviewed by Glick, J. Ind. Microbiol. 1: 277 (1987); Watson et al.,MOLECULAR BIOLOGY OF THE GENE, 4th Ed., Benjamin Cummins (1987); Ausubelet al., supra, and Sambrook et al., supra.

The invention also provides for a host cell carrying the nucleic acidsof the invention. A preferred prokaryotic host is E. coli. Preferredstrains of E. coli include Y1088, Y1089, CSH18, ER1451, and ER1647 (see,for example, Brown (Ed.), MOLECULAR BIOLOGY LABFAX, Academic Press(1991)). An alternative preferred host is Bacillus subtilus, includingsuch strains as BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA CLONING: A PRACTICAL APPROACH,Glover (Ed.), IRL Press (1985)). Other host include mammalian cells.

Methods for expressing nucleic acids are well-known to those of skill inthe art. See, for example, Ward et al., “Genetic Manipulation andExpression of Antibodies,” in MONOCLONAL ANTIBODIES: PRINCIPLES ANDAPPLICATIONS, pages 137-185 (Wiley-Liss, Inc. 1995). Moreover,expression systems for cloning antibodies in prokaryotic cells arecommercially available. For example, the IMMUNO ZAP.TM. Cloning andExpression System (Stratagene Cloning Systems; La Jolla, Calif.)provides vectors for the expression of antibody light and heavy chainsin E. coli.

The nucleic acids of the invention is preferably expressed in eukaryoticcells, and especially mammalian, insect, and yeast cells. Especiallypreferred eukaryotic hosts are mammalian cells. Mammalian cells providepost-translational modifications to the cloned polypeptide includingproper folding and glycosylation. For example, such mammalian host cellsinclude COS-7 cells (ATCC CRL 1651), non-secreting myeloma cells(SP2/0-AG14; ATCC CRL 1581), rat pituitary cells (GH₁; ATCC CCL 82), andrat hepatoma cells (H-4-II-E; ATCC CRL 1548).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, and simian virus. In addition, promoters from mammalianexpression products, such as actin, collagen, or myosin, can beemployed. Alternatively, a prokaryotic promoter (such as thebacteriophage T3 RNA polymerase promoter) can be employed, wherein theprokaryotic promoter is regulated by a eukaryotic promoter (for example,see Zhou et al., Mol. Cell. Biol. 10:4529 (1990); Kaufinan et al., Nucl.Acids Res. 19:4485 (1991)). Transcriptional initiation regulatorysignals may be selected which allow for repression or activation, sothat expression of the genes can be modulated. In general, eukaryoticregulatory regions will include a promoter region sufficient to directthe initiation of RNA synthesis. Such eukaryotic promoters include thepromoter of the mouse metallothionein I gene (Hamer et al., J. Mol.Appl. Gen. 1:273 (1982)); the TK promoter of Herpes virus (McKnight,Cell 31:355 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304 (1981)); the Rous sarcoma virus promoter (Gorman etal., supra); the cytomegalovirus promoter (Foecking et al., Gene 45:101(1980)); the yeast gal4 gene promoter (Johnston, et al., Proc. Natl.Acad. Sci. (USA) 79:6971 (1982); Silver et al., Proc. Natl. Acad. Sci.(USA) 81:5951 (1984)); and the IgG promoter (Orlandi et al., Proc. Natl.Acad. Sci. USA 86:3833 (1989)).

Strong regulatory sequences are the most preferred regulatory sequencesof the present invention. Examples of such preferred regulatorysequences include the SV40 promoter-enhancer (Gorman, “High EfficiencyGene Transfer into Mammalian cells,” in DNA CLONING: A PRACTICALAPPROACH, Volume II, Glover (Ed.), IRL Press pp. 143-190 (1985)), thehCMV-MIE promoter-enhancer (Bebbington et al., Bio/Technology 10:169(1992)), and antibody heavy chain promoter (Orlandi et al., Proc. Natl.Acad. Sci. USA 86:3833 (1989)). Also preferred are the kappa chainenhancer for the expression of the light chain and the IgH enhance(Gillies, “Design of Expression Vectors and Mammalian Cell SystemsSuitable for Engineered Antibodies,” in Antibody Engineering: APractical Guide, C. Borrebaeck (Ed.), W. H. Freeman and Company, pp.139-157 (1992); Orlandi et al., supra).

The PPC sequence and an operably linked promoter may be introduced intoeukaryotic cells as a non-replicating DNA molecule, which may be eithera linear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replication,the expression of the protein may occur through the transient expressionof the introduced sequence. Preferably, permanent expression occursthrough the integration of the introduced sequence into the hostchromosome.

Preferably, the introduced sequence will be incorporated into a plasmidor viral vector that is capable of autonomous replication in therecipient host. Several possible vector systems are available for thispurpose. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired genomic or cDNA sequences into the host chromosome.

Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,Mol. Cell. Biol. 3:280 (1983), Sambrook et al., supra, Ausubel et al.,supra, Bebbington et al., supra, Orlandi et al., supra, and Fouser etal., Bio/Technology 10:1121 (1992); Gillies, supra. Genomic DNAexpression vectors which include intron sequences are described byOrlandi et al., supra. Also, see generally, Lemer et al. (Eds.), NEWTECHNIQUES IN ANTIBODY GENERATION, Methods 2(2) (1991).

Methods Involving Targetable Constructs

The invention also provides for a method of treating or diagnosing adisorder. In the method, an PPC of the invention which has at least (A)one ABS specific for an epitope of a targeted tissue and (B) one ABSspecific for a targetable construct is provided is administered to thepatient. Following the administration of the PPC, the targetableconstruct is administered to the patient. The PPC and the targetableconstruct may be administered to the patient at substantially the sametime.

The targetable construct, for the purposes of this disclosure may be oftwo formulas.

In the first structure, the targetable construct is a compound of theformula:X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH₂;

-   -   where the compound includes a hard acid cation chelator at X or        Y, and a soft acid cation chelator at remaining X or Y; and        wherein the compound further comprises at least one diagnostic        or therapeutic cation, and/or one or more chelated or chemically        bound therapeutic agent, diagnostic agent, or enzyme (described        elsewhere in this disclosure). The diagnostic agent could be,        for example, Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II),        Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III), V(IV) ions        or a radical.

In the second formula, the targetable construct is a compound of theformula:X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH₂;

-   -   where the compound includes a hard acid cation chelator or a        soft acid cheator at X or Y, and nothing at the remaining X or        Y; and wherein the compound further comprises at least one        diagnostic or therapeutic cation, and/or one or more chelated or        chemically bound therapeutic agent, diagnostic agent, or enzyme        (described elsewhere in this disclosure). The diagnostic agent        could be, for example, Gd(III), Eu(III), Dy(III), Pr(III),        Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II),        Ti(III), V(IV) ions or a radical.

Any method of the invention that uses a targetable construct may alsouse a combination of targetable constructs. In a preferred embodiment,the targetable constructs are IMP241, IMP281 (FIG. 9A), IMP284 (FIG.9B), IMP288, or a combination thereof.

In this method, a clearing composition may be optionally administered tothe patient to clear non-localized PPC from circulation. The clearingcompound is administered after the administration of the PPC but beforethe administration of the targetable construct. These methods aredescribed in detail in U.S. Pat. No. 4,624,846, WO 92/19273, and Sharkeyet al., Int. J. Cancer 51: 266 (1992).

The described method may be used for in vivo diagnosis. The method ofdiagnostic imaging with radiolabeled monoclonal antibodies is well-knownand is applicable for the PPC of this invention. In the technique ofimmunoscintigraphy, for example, antibodies are labeled with aγ-emitting radioisotope and introduced into a patient. A γ camera isused to detect the location and distribution of γ-emittingradioisotopes. See, for example, Srivastava (ed.), RADIOLABELEDMONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press 1988),Chase, “Medical Applications of Radioisotopes,” in REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 990), Brown, “Clinical Use of MonoclonalAntibodies,” in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al. (eds.)(Chapman & Hall 1993), and Goldenberg, CA—A Cancer Journal forClinicians 44: 43 (1994). The methods of the invention may be practiced,for example, by the substitution of the monoclonal antibodies of theabove referenced techniques with the PPCs of the invention.

For diagnostic imaging, radioisotopes may be bound to a PPC eitherdirectly, or indirectly by using an intermediary functional group.Useful intermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see U.S. Pat. No. 5,057,313 and U.S. Pat. No. 5,128,119.

For purely diagnostic purposes (as opposed to therapeutic ordiagnostic/therapeutic purposes) radiation dose delivered to the patientis maintained at as low a level as possible by choosing an isotope withthe best combination of minimum half-life, minimum retention in thebody, and minimum quantity of isotope which will permit detection andaccurate measurement. Examples of radioisotopes that can be bound to thePPC and are appropriate for diagnostic imaging include γ-emitters andpositron-emitters such as ⁹⁹Tc, ⁶⁷Ga, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,⁵¹Cr, ⁸⁹Zr, ¹⁸F and ⁶⁸Ga. Other suitable radioisotopes are known tothose of skill in the art. Preferred γ-emitters have a γ radiationemission peak in the range of 50-500 Kev, primarily because the state ofthe art for radiation detectors currently favors such labels. Examplesof such γ-emitters include ⁹⁹Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.

The PPCs also can be labeled with paramagnetic ions for purposes of invivo diagnosis. Elements that are particularly useful for magneticresonance imaging include Gd, Mn, Dy and Fe ions. Other methods forenhancing in vivo diagnosis may be found, for example, in U.S. Pat. Nos.6,096,089, 5,965,131 and 5,958,048.

In an alternate approach, detection methods are improved by takingadvantage of the binding between avidin/streptavidin and biotin. Avidin,found in egg whites, has a very high binding affinity for biotin, whichis a B-complex vitamin. Streptavidin, isolated from Streptomycesavidinii, is similar to avidin, but has lower non-specific tissuebinding and therefore, streptavidin often is used in place of avidin. Abasic diagnostic method comprises administering a PPC compositeconjugated with avidin/streptavidin (or biotin), injecting a clearingcomposition comprising biotin (or avidin/streptavidin), andadministering a conjugate of a diagnostic agent and biotin (oravidin/streptavidin). Preferably, the biotin (or avidin/streptavidin)component of the clearing composition is coupled with a carbohydratemoiety (such as dextran) or a polyol group (e.g., polyethylene glycol)to decrease immunogenicity and permit repeated applications.

A modification of the basic method is performed by parenterallyinjecting a mammal with a PPC which has been conjugated withavidin/streptavidin (or biotin), injecting a clearing compositioncomprising biotin (or avidin/streptavidin), and parenterally injecting apolyspecific PPC according to the present invention, which furthercomprises avidin/streptavidin (or biotin). See WO 94/04702.

In a further variation of this method, improved detection can beachieved by conjugating multiple avidin/streptavidin or biotin moietiesto a polymer which, in turn, is conjugated to a PPC component. Adaptedto the present invention, monospecific or polyspecific PPCs can beproduced which contain multiple avidin/streptavidin or biotin moieties.Techniques for constructing and using multiavidin/multistreptavidinand/or multibiotin polymer conjugates to obtain amplification oftargeting are disclosed by Griffiths, PCT application numberPCT/US94/04295.

In another variation, improved detection is achieved by injecting atargeting PPC composite conjugated to biotin (or avidin/streptavidin),injecting at least one dose of an avidin/streptavidin (or biotin)clearing agent, and injecting a diagnostic composition comprising aconjugate of biotin (or avidin/streptavidin) and a naturally occurringmetal atom chelating protein which is chelated with a metal detectionagent. Suitable targeting proteins according to the present inventionwould be ferritin, metallothioneins, ferredoxins, and the like. See,PCT/US94/05149.

In another embodiment, the methods of the invention may be used fortherapy. In the therapeutic methods, a suitable therapeutic agent isselected from the group consisting of radioisotope, boron addend,immunomodulator, toxin, photoactive agent or dye, cancerchemotherapeutic drug, antiviral drug, antifungal drug, antibacterialdrug, antiprotozoal drug and chemosensitizing agent (See, U.S. Pat. Nos.4,925,648, 4,932,412). Suitable chemotherapeutic agents are described inREMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in Goodman and Gilman's THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co., New York,1980 and 2001 editions). Moreover a suitable therapeutic radioisotope isselected from the group consisting of α-emitters, β-emitters,.γ.-emitters, Auger electron emitters, neutron capturing agents thatemit α-particles and radioisotopes that decay by electron capture.Preferably, the radioisotope is selected from the group consisting of²²⁵Ac, ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ¹⁷⁷Lu, ²¹³Bi,¹⁰B, and ²¹¹At.

Boron, when used as a therapeutic agent is useful in boron neutroncapture therapy (BNCT). BNCT is based on the nuclear reaction whichoccurs when a stable isotope, B-10 (present in 19.8% natural abundance),is irradiated with thermal neutrons to produce an a particle and a Li-7nucleus. These particles have a path length of about one cell diameter,resulting in high linear energy transfer. Just a few of the short-range1.7 MeV α particles produced in this nuclear reaction are sufficient totarget the cell nucleus and destroy it. Barth et al., Cancer, 70:2995-3007 (1992). Since the ¹⁰B(n,α)₇ Li reaction will occur, andthereby produce significant biological effect, only when there is asufficient number of thermal neutrons and a critical amount of B-10localized around or within the malignant cell, the radiation produced islocalized. The neutron capture cross section of B-10 far exceeds that ofnitrogen and hydrogen found in tissues, which also can undergo capturereactions, (relative numbers: 1 for N-14, 5.3 for H-1, and 11560 forB-10), so that once a high concentration differential of B-10 isachieved between normal and malignant cells, only the latter will beaffected upon neutron irradiation. This is the scientific basis forboron neutron capture therapy. This method is described in more detailin Barth et al., supra; Barth et al. Cancer Res., 50: 1061-70 (1990);Perks et al., Brit. J. Radiol., 61: 1115-26 (1988).

Therapeutic preparations contemplated herein comprise PPC comprising anABS specific for an epitope of a pathogen. This PPC is conjugated to atherapeutically effective radioisotope and/or antibiotic/cytotoxic drug,in a suitable vehicle for parenteral administration. A therapeuticpreparation may likewise comprise a polyspecific anti-pathogen PPCcomposite conjugated to a radioisotope and/or antibiotic/cytotoxic drug.

It is advantageous in certain cases to combine a drug with aradionuclide, especially where the pathogen “hides” or is somewhatinaccessible. The longer range action of radionuclides can reach hiddenpathogen so long as some antigen is accessible to the conjugate. Also,radiation can cause lysis of an infected cell and expose intracellularpathogen to the antimicrobial drug component of the conjugate.

The anti-microbial polyspecific imaging PPCs and monospecific orpolyspecific therapeutic PPCs according to the invention also can beconveniently provided in a therapeutic or diagnostic kit for PPCtargeting to a focus of infection. Typically, such a kit will comprise avial containing the PPC conjugate of the present invention, either as alyophilized preparation or in an injection vehicle. If the conjugate isto be used for scintigraphic imaging or for radioisotope therapy, itwill generally be provided as a cold conjugate together with reagentsand accessories for radiolabeling, in separate containers, while MRIagents and therapeutic drug/toxin conjugates will generally be suppliedwith a paramagnetic species or an antibiotic/cytotoxic agent alreadyconjugated to the PPC. The kit may further contain a second, separatelypackaged, unlabeled PPC specific the therapeutic agent, a carriertherefor, or a chelating agent for the radionuclide or paramagnetic ion.

It is well known in the art that various methods of radionuclide therapycan be used for the treatment of cancer and other pathologicalconditions, as described. e.g., in Harbert, “Nuclear Medicine Therapy”,New York, Thieme Medical Publishers, 1087, pp. 1-340. A clinicianexperienced in these procedures will readily be able to adapt thecytokine adjuvant therapy described herein to such procedures tomitigate the hematopoietic side effects thereof. Similarly, therapy withcytotoxic drugs, either administered alone or as PPC conjugates for moreprecisely targeted therapy. e.g., for treatment of cancer, infectious orautoimmune diseases, and for organ rejection therapy, is governed byanalogous principles to radioisotope therapy with isotopes orradiolabeled antibodies. Thus, the ordinary skilled clinician will beable to adapt the description of cytokine use to mitigate marrowsuppression and other such hematopoietic side effects by administrationof the cytokine before, during and/or after drug therapy.

Therapeutically useful immunoconjugates can be obtained by conjugatingphotoactive agents or dyes to a PPC of the invention. Fluorescent andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy (Jori et al.,(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (LibreriaProgetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. Mew et al., J. Immunol. 130:1473 (1983); idem.,Cancer Res. 45:4380 (1985); Oseroffet al., Proc. Natl. Acad. Sci. USA83:8744 (1986); idem., Photochem. Photobiol. 46:83 (1987); Hasan et al,Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med.9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991). The presentinvention contemplates the therapeutic use of PPC comprising photoactiveagents or dyes. Anti-CD 19 and anti-CD20 antibodies are known to thoseof skill in the art. See, for example, Ghetie et al, Cancer Res. 48:2610(1988); Hekrnan et al., Cancer Immunol. Immunother. 32:364 (1991);Kaminski et al., N. Engl. J. Med. 329:459 (1993); Press et al., N. Engl.J. Med. 329:1219 (1993); Maloney et al., Blood 84:2457 (1994); Press etal., Lancet 346:336 (1995); Longo, Curr. Opin. Oncol. 8:353 (1996).

The targetable construct may contain ¹⁰B atoms. In this case, the methodmay comprise an additional step of effecting BNCT of a diseased tissue(including neoplastic tissue) where the targetable construct is located.See U.S. Pat. Nos. 5,846,741, 6,228,362 for a discussion of BNCT.

In another embodiment of the invention, the targetable construct of themethod may comprise an enzyme. The enzyme may be one that can increasethe cytotoxicity of a drug. For example, the enzyme may convert a drugfrom a nontoxic form to a toxic form. Alternatively, the enzyme mayconvert a toxic drug to an even more toxic drug. Examples of suchenzyme-prodrug binding partners are I-131-antibody-carboxypeptidase G2and topoisomerase-inhibiting prodrug CPT-11; β-lactamase andcephalosporin-doxorubicin; alkaline phosphatase and etoposide phosphate;carboxypeptidase G2 and glutamic acid derivative of benzoic acidmustard; and β-glucuronidase and the glucuronide of any drug which canform a glucuronide, such as p-hydroxyaniline mustard. Other examples oftargeted enzymes for prodrug activation are discussed in BioconjuateChem., Vol. 4, (1), 3-9 (1993).

In the methods where the PPC is labeled or the targetable construct islabeled, the method may be used to detect a target cell, target tissue,or a pathogen (i.e., infectious agent) in a patient.

Methods for Producing PPC

The invention also provides for methods for producing the PPC of theinvention. In one embodiment, the methods comprise providing a firstpolypeptide having an amino acid sequence comprising 3 or 4 v-regions(i.e., a₁, a₂, a₃, etc.) linearly arranged in the polypeptide sequence,optionally comprising amino acid linking sequences interspersed betweenthe v-regions, and providing a second polypeptide having an amino acidsequence comprising 3 or 4 v-regions (i.e., b₁, b₂, b₃, etc.) linearlyarranged in the polypeptide sequence, optionally comprising amino acidlinking sequences interspersed between the v-regions; and contacting thefirst and second polypeptides under appropriate conditions such that theindividual polypeptide chains arrange laterally to one another and bindto one another by the complemetarity binding of corresponding v-regions(i.e. a₁ to b₁, a₂ to b₂, a₃ to b₃, etc.) to form the PPC.

The methods of producing the PPC's may be performed, for example, byproducing the polypeptides on a peptide synthesizer and combining themin solution under appropriate conditions to allow for thecomplementarity binding of the individual polypeptide chains. Those ofordinary skill in the art are aware of several such methods forcombining the individual polypeptide chains. For example polypeptide 1and polypeptide 2 of a PPC may be synthesized on a peptide synthesizerusing following the manufacturer's instruction (e.g., AppliedBiosystems). Alternatively, peptides may be ordered by mail from acommercial laboratory (e.g., Sigma-Genosys, The Woodlands, Tex.). Thedried peptides may be mixed and solubilized in water or water with 5%NH₄OH to produce the PPC.

In a preferred embodiment, the two PPC polypeptides may be coexpressingin a host cell. For example, an expression plasmid (referred to hereinas the coexpression plasmid) that can co-express two different genesinserted into two different cloning sites may be chosen (e.g., BS14HP-GAP+). Nucleic acid molecules with open reading frames that encodepolypeptide 1 and polypeptide 2 of PPC may be cloned into the twocloning sites. The nucleic acid molecules may be cloned by traditionaltechniques or they may be synthesized using an oligonucleotidesynthesizer. The coexpression plasmid may be transfected into aeukaryotic host such as a yeast cell for expression. PPC may be producedby culturing the eukaryotic host cell culture until a desired quantityof PPC is produced.

In any of the production methods of the invention, the produced PPC maybe a tagged PPC. Tagged PPC may comprise an additional peptide sequence,such as the FLAG sequence or the polyHIS sequence. This sequence wouldallow any expressed PPC to be purified with the proper affinity column.

Another example of a suitable expression system for diabodies andtriabodies (which includes the PPC of this invention) is the pdHL2vector, which has an amplifiable murine dhfr gene that allows subsequentselection and amplification by methotrexate treatment. Gillies et al.,J. Immunol. Methods 125:191 (1989). The pdHL2 vector providesindependent expression of two genes that are independently controlled bytwo metallothionine promoters and IgH enhancers. One example of using anamplifiable selectable marker to increase expression in a mammalianrecombinant host cell line is shown in Example 2.

Suitable host cells or cell lines for the expression of the PPC of theinvention are known to one of skill in the art and are also listed inthe definition of “host cell” above. One host cell is a human cell—whichwould enable any expressed molecules to be modified with humanglycosylation patterns. It should be noted that there is no indicationthat a human host cell is essential or preferred for the methods of theinvention.

As an illustration, SP2/0 cells can be transfected by electroporationwith linearized pdHL2 vector that contains coding sequences for twoantibody components. Selection can be initiated 48 hours aftertransfection by incubating cells with medium containing 0.05-0.1 μMmethotrexate. Amplification of the two antibody sequences is achieved bya stepwise increase in methotrexate concentration up to 5 μM.

To ensure that the PPC was formed correctly, or for purification, thePPC may be purified by an antigen affinity column which is loaded withan antigen that is recognized by an ABS of the PPC. An antigen affinitypurification column can purify only those PPC with properly formed ABSbecause PPC without the proper ABS should not bind to the affinitycolumn matrix. The antigen affinity purification may be performedmultiple times. For example, if the ABSs of a PPC recognize antigen 1,antigen 2 and antigen 3, the PPC may be purified by three antigenaffinity purification columns each loaded with one of the threeantigens. Other methods of purification, such as, for example,precipitation of proteins, size exclusion chromatography,co-precipitation and co renaturation are known to those of skill in theart.

The following examples are provided to illustrate, but not to limit, theclaimed invention.

EXAMPLES Example 1 BS14HP, a Bispecific Trivalent Heterodimer

Design.

BS14HP was designed for the constitutive expression of foreign genes inPichia pastoris using the GAP promoter system. Transfection of P.pastoris cells with a linearized DNA plasmid (BS14HP-GAP+) results inthe stable and site-specific integration of the two DNA segments (FIG.1A) into the GAP locus of the host's chromosome. These two DNA segmentscontain open reading frames, SEQ ID NO:1 and SEQ ID NO:2, which codesfor polypeptide 1 (SEQ ID NO:9) and polypeptide 2 (SEQ ID NO:10)respectively. As each of the two DNA segments also contains nucleotidesequences for the GAP promoter, two mRNA species that encode the aminoacid sequences of polypeptide 1 and polypeptide 2 are synthesized in thesame host cell.

Polypeptide 1.

α-Factor-h679V_(H)-GGGGS-hMN-14V_(K)-LEGGGS-hMN-14V_(H)-6His (SEQ IDNO:1)

Polypeptide 2.

α-Factor-hMN-14V_(K)-GGGQFM-hMN-14V_(H)-GGGGS-h679V_(K)-6His (SEQ IDNO:2)

The “α-factor,” as shown in the schematic of polypeptide 1 and 2 above,represents is a signal peptide that is removed during synthesis andprotein transport, which resulting in secretion of the protein (withoutthe signal peptide) into the media. The carboxyl terminal hexa-histadine(6His) sequence allows for rapid and efficient purification of thesecreted protein with commercially available immobilized metal affinitychromatography (IMAC) material. hMN-14 V_(H) represents the amino acidsequence of the variable region of the heavy chain of (V_(H) region) ahumanized monoclonal antibody (Mab) that binds specifically tocarcinoembryonic antigen (CEA; Shevitz et al, J. Nucl. Med., suppl., 34,217P, 1993). h679VK represents the humanized murine monoclonal antibodydesignated 679 (an antibody of the IgG1, kappa class) binds with highaffinity to molecules containing the tri-peptide moietyhistamine-succinyl-glycyl (referred to herein as “HSG”; Morel et al,Molecular immunology, 27, 995-1000, 1990). The nucleotide sequencepertaining to the variable domains (V_(H) and V_(K)) of 679 has beendetermined (Qu et al, unpublished results). Humanized versions of the679 variable domains (Rossi. et al, unpublished results) were used inthe design of this construct.

The short peptide linkers, GGGGS, LEGGGS, GGGQFM, and GGGGS, between thevariable domains in the constructs are designed to discourageintra-polypeptide domain pairing. It is anticipated that the twodifferent polypeptides (FIG. 1B) would associate with each othernoncovalently by pairing the cognate V_(H) and V_(K) domains and therebyforming two functional binding sites for CEA and one functional bindingsite for HSG as shown in FIG. 1C.

PGAPZα+ Modified Vector

The novel construct pGAPZα+, depicted in FIG. 2, was engineered to makebispecific constructs through the synthesis of two heterologouspolypeptides from a single Pichia host cell. Two overlappingoligonucleotides, which constitute the SS1 linker, were synthesized,phosphorylated with T4 polynucleotide kinase and annealed by heating to95° C. and then slowly cooling to room temperature over 30 minutes. SS1Linker Top 5′- gatcccctgc agggagctca ctagta -3′ (SEQ ID NO:3) SS1 Linkerbottom 5′- gatctactag tgagctccct gcaggg -3′ (SEQ ID NO:4)

The oligonucleotide duplex was ligated into the BamHI site of thepGAPZαA vector (Invitrogen) and transformants were screened for thepresence of the linker in the proper orientation

Construction of the Pichia Expression Plasmid BS14HP-GAP+

Cloning of BS14-Orf-1-pGAPzα+

Using the plasmid construct hMN-14V_(H)-L5-h679VK-GAP+(Rossi et al,unpublished results) as a template, a PCR reaction was performed togenerate the amplimer XhoI-L6-hMN-14V_(H)-SalI using the followingoligonucleotide primers: L6-hMN14VH Xho Left5′-catactcgagggcggaggtagcgaggtccaactggtggagagc-3′ (SEQ ID NO:5)hMN14V_(H) SalI Right 5′- cttagtcgacggagacggtgaccggggtc -3′ (SEQ IDNO:6)

The PCR amplimer was cloned into pGemT vector (Promega) and screened forclones inserted in the 5′-T7 orientation. This construct,L6-hMN-14-pGemT(T7), was digested with NcoI and XhoI restriction enzymesand ligated with a DNA fragment containing h679V_(H)-L5-hMN14V_(K) thatwas excised from the h679V_(H)-L5-hMN-14VK-GAP+ plasmid construct (Rossiet al, unpublished results) with NcoI and XhoI restriction enzymes togenerate the construct to generate the staging plasmid constructBS14HPorf1-pGemT. This staging construct was first digested with NcoIrestriction endonuclease and the ends were made blunt by filling withthe Klenow fragment of the DNA polymerase. Following the Klenow fragmenttreatment, the DNA molecule was digested with SalI restrictionendonuclease to generate a fragment named BS14HP-orf1. The pGAPzα+vector (FIG. 2) was first digested with EcORI restriction endonucleaseand the ends were made blunt by filling with Klenow enzyme, and then itwas digested with SalI. The digested vector was ligated with the insertfragment to generate BS14orf1-pGAPZα+.

Cloning of BS14-orf2-PGAPZα+AVRX

A PCR reaction was performed to generate the amplimerEcoRI-L5-hMN-14V_(K)-L5-MfeI using the plasmid constructh679VH-L5-hMN-14VK-GAP+ (Rossi et al, unpublished results) as a templateand the following primers: HMN-14VK EcoRI Left 5′-ctaggaattc gacatccagctgacccagag-3′ (SEQ ID NO:7) hMN14V_(K)-L5 MfeI Right 5′-cgtacaattggccacctcca cgtttgattt ccaccttgg-3′ (SEQ ID NO:8)

The amplimer was digested with EcoRI and MfeI restriction enzymes andligated with the plasmid construct hMN-14VH-L5-h679VK-AvrX (Rossi et al,unpublished results) that was digested with EcoRI to generate theconstruct BS14HP-orf2-pGAPZα+AVRX.

Final Assembly of BS14HP-GAP+

The construct BS14HP-orf2-pGAPZα+AVRX was digested with NsiI and SpeIand separated by agarose gel electrophoresis. A 2260 bp DNA fragmentcontaining the BS14-orf2 coding sequence was isolated from the agarosegel. This isolated nucleic acid molecule was digested with SbfI and SpeIrestriction endonuclease and ligated with the NsiI/SpeI BS14-orf2fragment (discussed above) to generate the final construct BS14HP-GAP+

Constitutive Expression of BS14HP in Pichia Pastoris

The BS14HP-GAP+ construct was prepared for transfection by digestionwith AvrII restriction endonuclease. This linearized construct was usedto transfect the X-33 strain of Pichia pastoris by electroporation usingstandard methods. Stable transfectants were isolated on YPD-agar platescontaining 100g/ml of zeocin. Nine zeocin-resistant colonies werere-streaked on YPD-zeocin plates and the isolated clones were used toinoculate baffled shake flasks containing modified YPD media (1% yeastextract, 2% tryptone, 2% dextrose, 0.4 μg/ml biotin, 1.34% yeastnitrogen base, 100 mM K₂HPO₄, pH 6.0). The flask cultures were shook at250 RPM and 30° C. for 48-72 hours to stationary phase where the opticaldensity at 600 nm was between 18 and 25. The media, which should containthe excreted recombinant protein, was clarified by centrifugation andassayed for active protein using a BIAcore sensorchip.

The analysis step is as follows. Samples of the culture media werediluted 1:5 in EB buffer (150 mM NaCl; 50 μM EDTA; 0.005% surfactantP20; 10 mM HEPES, pH 7.4) and injected (50 μl) over a high-density,HSG-coupled sensorchip in a BIAcoreX system. Following injection of thediluted media, EB containing 20 μg/ml of WI2 IgG, an anti idiotypicantibody to hMN-14, was injected (100111) over the sensorchip to confirmbispecific binding. The initial binding slopes were used to quantitateyields. Seven of the nine zeocin resistant clones tested producedbispecific protein with a yield of up to 3 mg per liter of culturemedia.

To purify a larger amount of recombinant protein, the culture media fromthe highest expressing clone was buffer exchanged by diafiltration intoNi Binding buffer (300 mM NaCl; 10 mM imidazole; 50 mM NaH₂PO₄, pH 8.0).Following buffer exchange, the media protein was loaded onto Ni-NTA IMACaffinity column. The column was washed extensively with buffercontaining 20 mM imidazole and eluded with a buffer containing 250 mMimidazole (250 mM imidazole; 50 mM NaCl; 25 mM Tris, pH 7.5). Theeluates were analyzed by BIAcore, as described above, and all of thebinding activity was retained (FIG. 3).

Biochemical Analysis of BS14HP

To assay the expressed and purified BS14HP, a protein sample wasanalyzed by reducing SDS-PAGE and visualized by Coomassie blue-stainedSDS-PAGE gel. The results, as shown in FIG. 4, showed that the samplesare were highly purified and lack significant protein contamination. Thepurified protein complex were resolved in the SDS-PAGE gel as comprisingtwo similar sized c(i.e., closely spaced bands) polypeptide chain thatmigrate in the gel at a rate that is near expected molecular weights of40,614 and 40,061 Daltons.

To further analyze the expressed product, the expressed protein wasanalyzed by MALDI TOF mass spectrometry and size exclusion HPLCanalysis. Both analysis of expressed BS14HP gave a single peakconsistent with an 81 kDa dimeric protein structure (FIG. 5).

The binding stoichiometry can be extrapolated from BIAcore sensorgramssuch as the one shown in FIG. 3. The WI2:BS14HP molar binding ratio canbe derived by comparison of the response units (RU) attained from BS14HPbinding to the HSG sensorchip with the further RU increase from WI2binding to BS14HP with normalization for the respective molecularweights. Several BS14HP preps were analyzed with the measured ratioranging from 0.8-0.9. This ratio indicates the presence of twofunctional CEA binding sites per molecule, as the theoretical maximumratio for such a protein is 1.0. As a comparison, a variety ofmonovalent CEA-binding bispecific (hMN-14×679) constructs all gave molarratios between 0.4 and 0.45.

The CEA binding of BS14HP was analyzed in a competitive ELISA andcompared to BS1.5H and hMN-14 F(ab′)₂, which have one and two CEAbinding groups, respectively (FIG. 6). HRP-conjugated hMN-14 IgG (1 nM)was mixed with either BS14HP, BS1.5H or hMN-14 F(ab′)₂ at concentrationsranging from 1 to 250 nM, prior to incubation in CEA-coated (0.5μg/well) wells. The IC₅₀ for BS14HP was 2.7 nM which is close to 2.0 nMfor hMN14 F(ab′)₂. The monovalent CEA binder, BS1.5H, had an IC₅₀ of 10nM. These results are consistent with the BIAcore analysis indemonstrating that BS14HP binds CEA divalently. Further, the bindingavidity is comparable to that of the native hMN-14 F(ab′)₂.

Confirmation that BS14HP has the ability to bind CEA bivalently wasprovided by SE-HPLC analysis of in vitro immunoreactivity. Whenincreasing amounts of CEA were mixed with ¹²⁵I-hBS14, two distinct peakshifts were evident by HPLC corresponding to complexes of 125I-hBS14bound to either one or two CEAs. (FIG. 6B).

In Vivo Analysis of BS14HP

The utility of BS14HP for tumor pretargeting was evaluated in GW-39tumor-bearing mice using a bivalent HSG peptide (IMP-241) labeled with¹¹¹In. IMP-241 has a structure of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2,where the N-terminal amino group of Phe is linked to a DOTA and theepsilon amino group of each lysine is derivatived with an HSG group. Thetetrapeptide backbone (Phe-Lys-(D-Tyr)-Lys) contains a d-amino acid(D-Tyr) and the carboxyl group of the C-terminal lys is amidated.

The results from this analysis were compared with those of chemicallylinked hMN-14×679 (Fab′×Fab′) and BS1.5H (bispecific diabody). Nude micebearing GW-39 (CEA positive) tumors were pre-targeted with BS14HP,BS1.5H or hMN-14×679. Initially, the bio-distribution was followed with¹³¹I-labeled bispecific agent. The tumor residence and blood clearanceof ¹³¹I-BS14HP is depicted in FIG. 7. As determined in preliminaryexperiments, the time interval between administration of bispecifictargeting agent and of ¹¹¹In-IMP241 peptide is the amount of timerequired for the former to clear the blood to a concentration of 1% ID/gor less. A pre-targeting clearance time of 24 hours was used for BS14HPand hMN-14×679. A 15-hour clearance time was used for the smallerBS1.5H, which clears the blood more rapidly. IMP-241 (Immunomedics,Inc), a peptide containing two HSG groups and a DOTA moiety, was loadedwith ¹¹¹Indium and injected in pre-targeted mice. The bio-distributionof the ¹¹¹In-IMP-241 was examined at 3 hours after injection (FIG. 8A)and the tumor/non-tumor ratios are shown in FIG. 8B.

The results indicate that approximately three-fold more ¹¹¹In-IMP241peptide was specifically bound to the tumor in mice pretargeted withBS14HP, as compared to mice pretargeted with either BS1.5H or hMN14×679.The radioactivity in all non-tumor organs was low and comparable amongstthe three pretargeting agents.

These experiments were performed again using polypeptide IMP281 (FIG.9A), IMP284 (FIG. 9B), and IMP288 (FIG. 9C) with the same results.

Example 2 hBS14, a Bispecific Trivalent Heterodimer Expressed in MyelomaCells

To demonstrate that similar PPCs could be made from other types of hostcell systems we developed a scheme for production of a fusion proteinnamed hBS14 in mammalian cell culture. The hBS14 PPC produced inmammalian cell culture was designed to be structurally and functionallysimilar to BS14HP, which was produced in the yeast P. pastoris (example1). A DNA plasmid vector was engineered for hBS14 expression and used togenerate transgenic cell lines in SP2/0-Ag14 mouse myeloma cells, NS0mouse myeloma, and YB2/0 rat myeloma cells. While these particular celllines were used, it is understood that the vectors of the invention maybe used in any mammalian cell lines, such as, for example, a human cellline.

Generation of an hBS14 DNA Expression Vector

The nucleic acid encoding the hBS14 polypeptides were recombinantlyinserted into the mammalian expression vector pdHL2, which permits theamplification of antibody production. The pdHL2 vector contains thegenes for IgG constant regions (CH an CK) and was originally designed toaccept variable domain cassettes and direct the synthesis of whole IgG.Since we are interested in expressing novel single chain-basedconstructs devoid of constant region sequences, it was necessary tocreate a new shuttle vector to facilitate the assembly and transfer ofthe hBS14 genes into the pdHL2 vector. See FIG. 10A.

Overlapping synthetic oligonucleotides (85mers) were annealed to formduplex DNA possessing the features shown in FIG. 10A. This duplex wasligated into the HindIII and EcoRI restriction endonuclease sites of thepGEM3z cloning vector (Promega) to generate the SV3 shuttle vector. Thevariable domain genes were amplified by PCR from BS14HP-GAP+(example 1)and assembled into open reading frames (ORFs) in the SV3 shuttle vectorvia the NcoI and SalI restriction endonuclease sites. SV3 constructswere generated for both ORF 1 and ORF 2, which encode polypeptides 1 and2 (See FIG. 10B).

Each ORF includes the IgG light chain leader peptide, which directssecretion of the nascent polypeptides, preceding the variable domaingenes, which are in turn followed by the codons for six histidines andtwo stop codons. The variable domains are separated by linker peptidesconsisting of 5 or 6 amino acid residues. ORF1 and ORF2 were sub-clonedinto a single pdHL2 expression vector. ORF1 was excised from its shuttlevector with Xba I and Bgl II restriction endonucleases and cloned intothe XbaI and BamHlI sites of pdHL2 to generate the intermediateconstruct hBS14ORF1-pdHL2. ORF2 was then excised from its shuttle vectorwith XhoI and EagI restriction enzymes and cloned into those same sitesof the intermediate hBS14ORF1-pdHL2 construct to generate the finaldi-cistronic expression vector hBS14-pdHL2 (FIG. 11).

Stable Transfection and Amplification of hBS14 Genes in SP2/0 MyelomaCells

SP2/0-Ag14 mouse myeloma cells have been used previously in conjunctionwith the pdHL2 expression vector for high-level expression ofrecombinant IgG. NS0 mouse myeloma and YB2/0 rat myeloma cells have beenused for high-level expression of recombinant IgG with other expressionvectors. The hBS14-pdHL2 DNA vector was linearized by digestion withEcoRI restriction endonuclease and successfully transfected into each ofthe three cell lines (4×10⁶ cells) by electroporation (450 volts, 25μF). The pdHL2 vector contains the gene for dihydrofolate reductase(DHFR) allowing clonal selection as well as gene amplification withmethotrexate (MTX).

Transfectants were cloned by plating in 96-well plates in the presenceof 0.05 μM MTX and the primary screening for hBS14-expressing clones wasaccomplished by ELISA. The ELISA screening format was as follows: Aconjugate consisting of an HSG-containing peptide (IMP239) and bovineserum albumin was first adsorbed to micro-plate wells and thenconditioned media from the putative clones were transferred to themicro-plate wells to allow hBS14 binding to the HSG groups of theconjugate. Bound hBS14 was detected with WI2, a rat anti-idiotype IgG tohMN-14, and HRP-conjugated goat anti-rat IgG. Several positive cloneswere identified and expanded. Expression of hBS14 was confirmed byBIAcore using an HSG (IMP239) sensorchip. An increase in response units(RU) following injection of culture media signified expression of hBS14.A further increase in RU with subsequent injection of WI2 demonstratedthat the hBS14 was bispecific and fully functional. With this method,standard concentration curves were generated using purified 679-proteinsallowing for accurate real time measurements of productivity. Theinitial productivity of the highest terminal culture hBS14 producer inSP2/0, YB2/0 and NS0 was 0.8 mg/L, 3.7 mg/L, and 4.4 mg/L, respectively.

Gene amplification and the resulting increase in productivity wereaccomplished by stepwise increase in MTX concentration in the culturemedia over several months. An example of the increase in productivity isshown for SP2/0 clone 1H6 in FIG. 12. The MTX concentration has beenincreased from 0.05 μM to 1 μM and the productivity has increased to 8mg/L, 16 mg/L and 9.3 mg/L for representative clones of SP2/0, YB2/0 andNS0, respectively without adverse effects. We expect these yields can befurther improved with further MTX treatment and selection. Typically,MTX concentrations can be increased up to 5 μM with significantadditional increase in productivity.

Production and Purification of hBS14

Nearly 100 mg of hBS14 has been purified to near homogeneity. Startingmaterial was generated in terminal roller bottle cultures of eachrepresentative cell line grown with 1 μM MTX. The purification processwas greatly facilitated by the generation of an HSG-based affinitypurification resin. Initial attempts using affigel-IMP239, the samepeptide that was used in both ELISA and BIAcore experiments, were lesssuccessful because the strong binding affinity made elution withoutprotein denaturation. None of the myriad elution buffers tested elutedthe hBS14 effectively. A new peptide (IMP291), which was designed tohave {fraction (1/10)} to {fraction (1/100)} lower affinity for 679, wassynthesized and conjugated to Affigel (BIO-RAD) by standard methods. Thehigh binding capacity (>20 mg/ml) Affigel-IMP291 proved to be ideal foraffinity purification of hBS14 by providing high yield, highpurification and high retention of activity.

Briefly, culture media from roller bottles was clarified by cross-flowmicrofiltration (0.2 μM) and then pH adjusted to 4.5 with citric acid.The hBS14 in the clarified and pH adjusted media was partly purifiedabout 25 fold by loading the media onto a S-sepharose cation exchangecolumn. The S-sepharose column was eluted with 2×PBS (0.3 M NaCl; 80 mMNaH₂PO₄, pH 7.4) and the eluate was loaded onto an Affigel-IMP291column. The column was eluted with 50 ml of 1M imidazole; 150 mMsucrose; 0.02% Tween-20; 50 mM Citrate, pH 4.5. The eluded product wasdialyzed into formulation buffer (150 mM Sucrose; 0.02% Tween-20; 10 mMNaAc, pH 4.5). This procedure allows elution of nearly 100% of the hBS14bound to the affinity column.

Biochemical Analysis of hBS14

Basic biochemical analysis demonstrated that the purification processresulted in highly purified hBS14. The native quaternary structure ofthe hBS14 was designed to be a 79.4 kDa heterodimer of polypeptide 1(39.94 kDa) and polypeptide 2 (39.5 kDa). The size exclusion HPLCprofile of purified hBS14 (Fig C) shows a major sharp peak with aretention time of 9.23 minutes, consistent with the profile of an 80 kDaprotein. BS1.5H diabody (54 kDa), hMN-14 triabody (78 kDa), and hMN-14F(ab′)₂ (100 kDa) were run in the same column as molecular weight andsize standards. These proteins had retention times of 9.60, 9.35, and8.77 minutes, respectively. The HPLC profiles are very similar amongbatches purified from each cell line (Fig C.). The peak at ˜11.4 minutesis a non-protein buffer peak. The small peak at 8.30 minutes constitutes3% of the total protein and is likely dimerized/aggregated hBS14.SDS-PAGE analysis was used to evaluate the purity and quality of thepolypeptide constituents of hBS14. The Coomassie blue-stained reducingSDS-PAGE gel shown in FIG. 14 demonstrates the high degree of purityachieved from this two-step purification process. Only trace amounts ofcontaminating protein were detected even when a lane was overloaded with4 μg of protein. This SDS-PAGE analysis indicates that the minor HPLCpeak (8.30 min) is indeed hBS14 aggregate and not contaminating protein.The molecular weights (MW) given for polypeptides 1 and 2 werecalculated from the deduced amino acid sequences of the polypeptides.The Mrs of the two bands are consistent with the calculated MW of thehBS14 polypeptides. As predicted, the two bands appear to be of equalintensity as they should be in equimolar concentration based on themolecular design. There is no evidence of appreciable proteindegradation. Isoelectric focusing (IEF) of the purified hBS14 shows amajor band near the isoelectric point (pI) of hBS14 (pI=7.73) ascalculated from the deduced amino acid sequence (FIG. 15). There aretrace bands at lower pI that are likely product related and may be theresult of negligible deamidation of some basic amino acid residues.Taken together, this combination of standard biochemical analysessuggests that the transgenic myeloma cells correctly synthesize andsecrete hBS14 as designed and that we have developed a robustpurification process capable of generating highly purified material. Thebiochemical properties of hBS14 were indistinguishable among batchesprepared from the different cell lines.

Functional characterization was provided by BIAcore experiments todemonstrate bispecific binding properties (FIG. 16). hBS14 bound tightlyto HSG that was immobilized on a sensorchip. The HSG-bound proteins wereable to capture subsequently added CEA or WI2, demonstrating that theycan simultaneously bind both antigens. If the WI2 binding is allowed toapproach saturation, the stoichiometry of the binding can be determined.The additional increase in RU resulting from WI2 binding was compared tothe initial RU increase of the hBS14 upon binding to the HSG-sensorchip.As each increase in RU level is directly proportional to the mass bound,the WI2:bsAb molar binding ratio can be calculated using the formula(RU_(WI2)/RU_(hBS14))×(MW_(hBS14)/MW_(WI2)). hBS14 was designed to bebivalent for CEA (and monovalent for HSG) and as such should bind WI2(also bivalent) with a 1:1 molar ratio. Indeed, the experimentallydetermined molar binding ratio of WI2 to hBS14 was found to be between0.7 and 0.8, approaching the theoretical maximum of 1.0. When equalconcentrations of hBS14 were bound to an HSG-sensorchip, BIAcoresensorgrams are indistinguishable between lots derived from either SP2/0or YB2/0 cells (FIG. 17).

The data demonstrate that the primary amino acid sequence is solelyresponsible for the structure and function of the PPC, independent ofthe host cell from which it is produced. The PPC hBS14 was not onlyequivalent among batches produced in three different mammalian celllines, it was also very similar with respect to structure and functionto BS14HP, which has similar primary amino acid sequences but isproduced in yeast.

Biopolymer Sequences:

The nucleic acid and amino acid sequence of the biopolymers used inExample 2 are as follows: hBS14 polypeptide 1 deduced amino acidsequence:MEVQLVESGGDLVKPGGSLKLSCAASGFTFSIYTMSWLRQTPGKGLEWVATLSGDGDDIYYPDSVKGR(SEQ ID NO:11)FTISRDNAKNSLYLQMNSLRAEDTALYYCARVRLGDWDFDVWGQGTTVSVSSGGGGSDIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRLEGGGSEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSVDHHHHHH orMetGluValGlnLeuValGluSerGlyGlyAspLeuValLysProGlyGlySerLeuLysLeuSerCy(SEQ ID NO:11)sAlaAlaSerGlyPheThrPheSerIleTyrThrMetSerTrpLeuArgGlnThrProGlyLysGlyLeuGluTrpValAlaThrLeuSerGlyAspGlyAspAspIleTyrTyrProAspSerValLysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCysAlaArgValArgLeuGlyAspTrpAspPheAspValTrpGlyGlnGlyThrThrValSerValSerSerGlyGlyGlyGlySerAspIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIleThrCysLysAlaSerGlnAspValGlyThrSerValAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrTrpThrSerThrArgHisThrGlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThrIleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGlnTyrSerLeuTyrArgSerPheGlyGlnGlyThrLysValGluIleLysArgLeuGluGlyGlyGlySerGluValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArgSerLeuArgLeuSerCysSerAlaSerGlyPheAspPheThrThrTyrTrpMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIleGlyGluIleHisProAspSerSerThrIleAsnTyrAlaProSerLeuLysAspArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuPheLeuGlnMetAspSerLeuArgProGluAspThrGlyValTyrPheCysAlaSerLeuTyrPheGlyPheProTrpPheAlaTyrTrpGlyGlnGlyThrProValThrValSerValAspHisHisHisHisHisHis hBS14 polypeptide 2 deduced amino acid sequence:MDIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGS(SEQ ID NO:12)GTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRGGGQFMEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSGGGGSDIVMTQSPSSLAVSPGERVTLTCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCTQVYYLCTFGAGTKLELKRLDHHHHHH. orMetAspIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIleTh(SEQ ID NO:12)rCysLysAlaSerGlnAspValGlyThrSerValAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrTrpThrSerThrArgHisThrGlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThrIleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGlnTyrSerLeuTyrArgSerPheGlyGlnGlyThrLysValGluIleLysArgGlyGlyGlyGlnPheMetGluValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArgSerLeuArgLeuSerCysSerAlaSerGlyPheAspPheThrThrTyrTrpMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIleGlyGluIleHisProAspSerSerThrIleAsnTyrAlaProSerLeuLysAspArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuPheLeuGlnMetAspSerLeuArgProGluAspThrGlyValTyrPheCysAlaSerLeuTyrPheGlyPheProTrpPheAlaTyrTrpGlyGlnGlyThrProValThrValSerGlyGlyGlyGlySerAspIleValMetThrGlnSerProSerSerLeuAlaValSerProGlyGluArgValThrLeuThrCysLysSerSerGlnSerLeuPheAsnSerArgThrArgLysAsnTyrLeuGlyTrpTyrGlnGlnLysProGlyGlnSerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyValProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleAsnSerLeuGlnAlaGluAspValAlaValTyrTyrCysThrGlnValTyrTyrLeuCysThrPheGlyAlaGlyThrLysLeuGluLeuLysArgLeuAspHisHisHisHisHisHis. hBS14 Openreading frame 1 (including coding sequence for the leader peptide)nucleic acid sequence:atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccatggaagtgca(SEQ ID NO:13)gctggtggagtcagggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtatttacaccatgtcttggcttcgccagactccgggaaaggggctggagtgggtcgcaaccctgagtggtgatggtgatgacatctactatccagacagtgtgaagggtcgattcaccatctccagagacaatgccaagaacagcctatatctgcagatgaacagtctaagggctgaggacacggccttgtattactgtgcaagggtgcgacttggggactgggacttcgatgtctggggccaagggaccacggtctccgtctcctcaggaggtggcggatccgacatccagctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggccagtcaggatgtgggtacttctgtagcttggtaccagcagaagccaggtaaggctccaaagctgctgatctactggacatccacccggcacactggtgtgccaagcagattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagcctccagccagaggacatcgccacctactactgccagcaatatagcctctatcggtcgttcggccaagggaccaaggtggaaatcaaacgtctcgagggcggaggtagcgaggtccaactggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctggcttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagtggattggagaaattcatccagatagcagtacgattaactatgcgccgtctctaaaggatagatttacaatatcgcgagacaacgccaagaacacattgttcctgcaaatggacagcctgagacccgaagacaccggggtctatttttgtgcaagcctttacttcggcttcccctggtttgcttattggggccaagggaccccggtcaccgtctcagtcgaccatcatcatcatcatcattga. HBS14Open reading frame 2 (including coding sequence for the leader peptide)nucleic acid sequence:atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccatggacatcca(SEQ ID NO:14)gctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggccagtcaggatgtgggtacttctgtagcctggtaccagcagaagccaggtaaggctccaaagctgctgatctactggacatccacccggcacactggtgtgccaagcagattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagcctccagccagaggacatcgccacctactactgccagcaatatagcctctatcggtcgttcggccaagggaccaaggtggaaatcaaacgtggaggtggccaattcatggaggtccaactggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctggcttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagtggattggagaaattcatccagatagcagtacgattaactatgcgccgtcgctaaaagatagatttacaatatcgcgagacaacgccaagaacacattgttcctgcaaatggacagcctgagacccgaagacaccggggtctatttttgtgcaagcctttacttcggcttcccctggtttgcttattggggccaagggaccccggtcaccgtctccggaggcggtggatccgacattgtgatgacacaatctccatcctccctggctgtgtcacccggggagagggtcactctgacctgcaaatccagtcagagtctgttcaacagtagaacccgaaagaactacttgggttggtaccagcagaaaccagggcagtctcctaaacttctgatctactgggcatctactcgggaatctggggtccctgatcgcttctcaggcagtggatccggaacagatttcactctcaccatcaacagtctgcaggctgaagacgtggcagtttattactgcactcaagtttattatctgtgcacgttcggtgctgggaccaagctggagctgaaacggctcgaccatcatcatcatcatcattga. Nucleic acid sequence ofhBS14-pDHL2 plasmid construct:ttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaaccc(SEQ ID NO:15)gacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaaggccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaagaattccgatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatctaaagccagcaaaagtcccatggtcttataaaaatgcatagctttaggaggggagcagagaacttgaaagcatcttcctgttagtctttcttctcgtagacttcaaacttatacttgatgcctttttcctcctggacctcagagaggacgcctgggtattctgggagaagtttatatttccccaaatcaatttctgggaaaaacgtgtcactttcaaattcctgcatgatccttgtcacaaagagtctgaggtggcctggttgattcatggcttcctggtaaacagaactgcctccgactatccaaaccatgtctactttacttgccaattccggttgttcaataagtcttaaggcatcatccaaacttttggcaagaaaatgagctcctcgtggtggttctttgagttctctactgagaactatattaattctgtcctttaaaggtcgattcttctcaggaatggagaaccaggttttcctacccataatcaccagattctgtttaccttccactgaagaggttgtggtcattctttggaagtacttgaactcgttcctgagcggaggccagggtcggtctccgttcttgccaatccccatattttgggacacggcgacgatgcagttcaatggtcgaaccatgagggcaccaagctagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagctcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctaactgacacacattccacagtcgactagaatatggatagtgggtgtttatgactctggataagcctgaacaattgatgattaatgcccctgagctctgttcttagtaacatgtgaacatttacttgtgtcagtgtagtagatttcacatgacatcttataataaacctgtaaatgaaagtaatttgcattactagcccagcccagcccatactaagagttatattatgtctgtctcacagcctgctgctgaccaatattgaaaagaatagaccttcgactggcaggaagcaggtcatgtggcaaggctatttggggaagggaaaataaaaccactaggtaaacttgtagctgtggtttgaagaagtggttttgaaacactctgtccagccccaccaaaccgaaagtccaggctgagcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcagccgaaactggagaggtcctcttttaacttattgagttcaaccttttaattttagcttgagtagttctagtttccccaaacttaagtttatcgacttctaaaatgtatttagaatttcgaccaattctcatgtttgacagcttatcatcgctgcactccgcccgaaaagtgcgctcggctctgccaaggacgcggggcgcgtgactatgcgtgggctggagcaaccgcctgctgggtgcaaaccctttgcgcccggactcgtccaacgactataaagagggcaggctgtcctctaagcgtcaccacgacttcaacgtcctgagtaccttctcctcacttactccgtagctccagcttcaccagatccctcgactctagacacaggccgccaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccatggaagtgcagctggtggagtcagggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtatttacaccatgtcttggcttcgccagactccgggaaaggggctggagtgggtcgcaaccctgagtggtgatggtgatgacatctactatccagacagtgtgaagggtcgattcaccatctccagagacaatgccaagaacagcctatatctgcagatgaacagtctaagggctgaggacacggccttgtattactgtgcaagggtgcgacttggggactgggacttcgatgtctggggccaagggaccacggtctccgtctcctcaggaggtggcggatccgacatccagctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggccagtcaggatgtgggtacttctgtagcttggtaccagcagaagccaggtaaggctccaaagctgctgatctactggacatccacccggcacactggtgtgccaagcagattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagcctccagccagaggacatcgccacctactactgccagcaatatagcctctatcggtcgttcggccaagggaccaaggtggaaatcaaacgtctcgagggcggaggtagcgaggtccaactggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctggcttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagtggattggagaaattcatccagatagcagtacgattaactatgcgccgtctctaaaggatagatttacaatatcgcgagacaacgccaagaacacattgttcctgcaaatggacagcctgagacccgaagacaccggggtctatttttgtgcaagcctttacttcggcttcccctggtttgcttattggggccaagggaccccggtcaccgtctcagtcgaccatcatcatcatcatcattgataagatcccgcaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcaggaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagagggagaagtgcccccacctgctcctcagttccagcctgaccccctcccatcctttggcctctgaccctttttccacaggggacctacccctattgcggtcctccagctcatctttcacctcacccccctcctcctccttggctttaattatgctaatgttggaggagaatgaataaataaagtgaatctttgcacctgtggtttctctctttcctcatttaataattattatctgttgttttaccaactactcaatttctcttataagggactaaatatgtagtcatcctaaggcgcataaccatttataaaaatcatccttcattctattttaccctatcatcctctgcaagacagtcctccctcaaacccacaagccttctgtcctcacagtcccctgggccatggtaggagagacttgcttccttgttttcccctcctcagcaagccctcatagtcctttttaagggtgacaggtcttacagtcatatatcctttgattcaattccctgagaatcaaccaaagcaaatttttcaaaagaagaaacctgctataaagagaatcattcattgcaacatgatataaaataacaacacaataaaagcaattaaataaacaaacaatagggaaatgtttaagttcatcatggtacttagacttaatggaatgtcatgccttatttacatttttaaacaggtactgagggactcctgtctgccaagggccgtattgagtactttccacaacctaatttaatccacactatactgtgagattaaaaacattcattaaaatgttgcaaaggttctataaagctgagagacaaatatattctataactcagcaattcccacttctaggggttcgactggcaggaagcaggtcatgtggcaaggctatttggggaagggaaaataaaaccactaggtaaacttgtagctgtggtttgaagaagtggttttgaaacactctgtccagccccaccaaaccgaaagtccaggctgagcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcagccgaaactggagaggtcctcttttaacttattgagttcaaccttttaattttagcttgagtagttctagtttccccaaacttaagtttatcgacttctaaaatgtatttagaatttcgaccaattctcatgtttgacagcttatcatcgctgcactccgcccgaaaagtgcgctcggctctgccaaggacgcggggcgcgtgactatgcgtgggctggagcaaccgcctgctgggtgcaaaccctttgcgcccggactcgtccaacgactataaagagggcaggctgtcctctaagcgtcaccacgacttcaacgtcctgagtaccttctcctcacttactccgtagctccagcttcaccagatccctcgagtctagacacaggccgccaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccatggacatccagctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggccagtcaggatgtgggtacttctgtagcttggtaccagcagaagccaggtaaggctccaaagctgctgatctactggacatccacccggcacactggtgtgccaagcagattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagcctccagccagaggacatcgccacctactactgccagcaatatagcctctatcggtcgttcggccaagggaccaaggtggaaatcaaacgtggaggtggccaattcatggaggtccaactggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctggcttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagtggattggagaaattcatccagatagcagtacgattaactatgcgccgtctctaaaggatagatttacaatatcgcgagacaacgccaagaacacattgttcctgcaaatggacagcctgagacccgaagacaccggggtctatttttgtgcaagcctttacttcggcttcccctggtttgcttattggggccaagggaccccggtcaccgtctccggaggcggtggatccgacattgtgatgacacaatctccatcctccctggctgtgtcacccggggagagggtcactctgacctgcaaatccagtcagagtctgttcaacagtagaacccgaaagaactacttgggttggtaccagcagaaaccagggcagtctcctaaacttctgatctactgggcatctactcgggaatctggggtccctgatcgcttctcaggcagtggatccggaacagatttcactctcaccatcaacagtctgcaggctgaagacgtggcagtttattactgcactcaagtttattatctgtgcacgttcggtgctgggaccaagctggagctgaaacggctcgaccatcatcatcatcatcattgataagatctcggccggcaagcccccgctccccgggctctcgcggtcgcacgaggatgcttggcacgtaccccgtctacatacttcccaggcacccagcatggaaataaagcacccaccactgccctgggcccctgcgagactgtgatggttctttccacgggtcaggccgagtctgaggcctgagtggcatgagggaggcagagcgggtcccactgtccccacactggcccaggctgtgcaggtgtgcctgggccgcctagggtggggctcagccaggggctgccctcggcagggtgggggatttgccagcgtggccctccctccagcagcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttt

Example 3 Affinity Purification of hBS14

hBS14 was purified to homogeneity using a novel affinity resin that wasprepared and used as described below.

Activation and Coupling of IMP291-affigel

IMP291 peptide (see structure in FIG. 18) was coupled to Affigel 102(BIO-RAD Laboratories, Hercules Calif.) using chloroacetic anhydride(CAA). CCA (1.5 g, 8.8 mmol) was dissolved in acetonitrile and added to30 ml of Affigel 102 slurry. The pH was adjusted to 9.0 withtriethylamine and reacted for 1 hour at room temperature to allowcoupling of CAA to amine groups on the Affigel. The CAA-Affigel waswashed and exchanged into 0.2M NaBorate, pH 8.0. A total of 166 mg ofIMP291 was dissolved in 10 ml of 0.2M NaBorate, pH 8.0 and then added tothe slurry, which was then rocked overnight at room temperature to allowcoupling of the peptide to the CAA-Affigel via thioether bond formation.The resin was quenched by adding cysteine in 0.2M NaBorate, pH 8.0 to afinal concentration of 20 mM and incubated for 1 hour at roomtemperature.

Qualification of the Affinity Resin

The IMP291-affigel resin was qualified as follows: A column was packedwith 0.5 ml of the resin and equilibrated with PBS. A total of 23.5 mgof BS1.5HP in 10 ml of PBS was passed over the column. A total of 14 mgof BS1.5HP was detected in the unbound fraction indicating 9.5 mg hadbound. A total of 9.2 mg was recovered in 7 ml of elution with 1MImidazole, 150 mM sucrose, 10 mM NaAc, pH 4.5. The binding capacity ofthe resin was determined to be 330 pmol/ml. For hBS14, this isequivalent to a capacity of 26.7 mg/ml.

Single-Step Purification of hBS14 with IMP291-Affigel

A total of 22 liters of hBS14 YB2/0 clone #8 roller bottle culturecontaining 144 mg of hBS14 (as estimated by BIAcore) was centrifuged andbrought to 2 mM EDTA, 0.02% Triton-X-100; and 10 mM Na₂HPO₄ Thesupernatant fluid was sterile-filtered through a 0.2 μM Millipak-200filter unit into an autoclaved 10-L bottle closed system. The filteredmedia was loaded over a 10 ml IMP291-affigel column (2.5 cm diameter) atflow rates ranging from 2 to 4 ml/min. The column was washed to baselinewith PBS and then eluted with 107 ml of elution buffer (1 M imidazole,150 mM sucrose, 50 mM citrate, pH 4.5). A total of 93 mg of hBS14 waseluted. Size exclusion HPLC, SDS-PAGE, IEF, and MALDI-TOF massspectrometry all indicated a highly purified homogeneous product fromthe single step IMP291-affigel affinity chromatography. BLAcore and invivo analysis demonstrated that the product was fully active.

Example 4 Use of hBS14 for Pre-Targeting of Human Colorectal TumorXenografts in Nude Mice

This example demonstrates the ability of the trivalent, bispecific hBS14molecule (hMN-14×hMN-14×679) to pre-target IMP-245, a ^(99m)Tc-labeledpeptide, to a human colonic tumor (GW-39) xenograft. The structure ofIMP 245 is shown in FIG. 19. IMP-245 was prepared and labeled usingstandard techniques known in the art. See, for example, publishedapplication US20030198595, which is hereby incorporated by reference inits entirety.

The experiment used 3 groups of 15 mice, each of which was necropsied,and 1 group of 5 mice that was imaged. Three groups of mice wereadministered 6 μCi ¹²⁵I-hBS14 (40 μg, 5.0×10⁻¹⁰ moles). In the lastgroup (imaging group), 3 mice received unlabeled hBS14. The amount of^(99m)Tc-IMP-245 administered to all the mice was ˜40 μCi (92 ng,5.0×10⁻¹¹ moles) for a bispecific:peptide ratio of 10:1. Mice werenecropsied at 1, 4, and 24 hours post-peptide administration, and weredivided into the following groups:

-   -   Group I: ¹²⁵I-hBS14 with 4-hr clearance followed by        ^(99m)Tc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and        24-hrs post-DCS injection]    -   Group II: ¹²⁵I-hBS14 with 24-hr clearance followed by        ^(99m)Tc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and        24-hrs post-DCS injection]    -   Group III: ¹²⁵I-hBS14 with 48-hr clearance followed by        ^(99m)Tc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and        24-hrs post-DCS injection]    -   Group IV: hBS14 with 48-hr clearance (3 mice) followed by        ^(99m)Tc-IMP-245 (all 5 mice) [5 mice; image mice at 1-, 3-, 6-,        and 24-hrs post-DCS injection]

Due to differences in tumor growth rates, only 20 mice were initiallyavailable for administration of hBS14. Fifteen mice were used to fillout Group II while 5 mice were used for Group I. The remaining mice(including the imaged mice) were injected one week later. Not all themice implanted with GW-39 tumors developed usable tumors and, therefore,Group I only had 10 mice and were sacrificed at 1 hr post-peptideinjection and 24 hrs post-injection.

The graph in FIG. 20 (top panel) shows the tumor uptake of the¹²⁵I-hBS14 and ^(99m)Tc-IMP-245 in mice when the hBS14 was given 4 hrsto clear prior to administration of peptide (Group I). At 1 hrpost-peptide administration there was 13.5±5.94% ID/g hBS14 in the tumorversus 2.9±0.46% ID/g of IMP-245 (4.7-fold less peptide than hBS14).After 24 hrs this ratio reversed with 2.4-fold more IMP-245 in the tumorversus the hBS14 (9.08±4.94% ID/g vs. 3.79±4.15% ID/g, respectively).Blood levels for the hBS14 and peptide were high at 1 hr post-injection(16.85±2.95% ID/g and 36.87+6.42% ID/g for hBS114 and IMP-245,respectively).

Data for the mice in Group II are shown in FIG. 20 (bottom panel)Approximately 2-fold more IMP-245 than hBS14 was observed in the tumors.The greatest amount of variation occurred in the 4 hr post-IMP245administration group (15.9±16.3% ID/g IMP-245 in the tumor). Thesedifferences do not appear to be due to hBS14 uptake since one mouse had4.6% ID/g hBS14 in its tumor and only 3.6% ID/g IMP-245 while anothermouse in this group also had 4.4% ID/g hBS14 but 18.1% ID/g IMP-245.There appears, however, to be a correlation between tumor size. Thesedata suggest that larger tumors have better targeting in mice.

The graph in FIG. 21 (top panel) shows the tumor uptake of ¹²⁵I-hBS14and ^(99m)Tc-IMP-245 in mice given 48 hrs to clear the hBS14 prior theadministration of the peptide (Group III). Like the Group II mice (24 hrhBS14 clearance), consistent targeting of the hBS14 to the tumor at allthree time-points was observed. At 1 hr post-peptide injection (49 hrspost-hBS14 administration) there was 3.50±0.86% ID/g hBS14 in the tumor.This level was maintained throughout two later time-points (52 hrs and72 hrs post-hBS14 administration) at 3.62±1.59% ID/g and 6.97±3.10%ID/g, respectively. These data suggest that the bivalent hMN-14 portionof the hBS14 molecule increased its ability to stay on the tumor withoutbeing shed or otherwise lost. This stabilized binding of hBS14 to thetumor also resulted in a relatively constant ^(99m)Tc-IMP-245 signal atthe tumor. At 1 hr post-peptide injection there was 21.03±2.47% ID/g atthe tumor. After 4 hrs there was 14.53±4.90% ID/g and 15.47±9.31% ID/gat 24 hrs post-injection. The differences between any of these threetime-points are not significany but, since ^(99m)Tc has such a shorthalf-life (6.02 hrs) the relative amount of actual signal in the tumorsat 24 hrs was 13-fold less.

The table shown in FIG. 22 summarizes the % ID/g of the ^(99m)Tc-IMP-245and the tumor to non-tumor ratios (T:NT) in the various tissues at 1 hrpost-peptide administration for all three groups of mice (4, 24, and 48hr hBS14 clearance). One hour post-peptide injection was used sinceearly time-points for imaging are clinically desirable.

The data from the imaged mice are shown in FIG. 23. The first pair ofimages shows the location of the tumors in the mice. hBS14 (5×10⁻¹⁰moles) was administered, followed after 48 hours by ^(99m)Tc-IMP-245 (40μCi; 5×10⁻¹¹ moles). Animals 1 & 2 were given peptide only, whileanimals 3, 4, & 5 were administered hBS14 followed by peptide. Theanimals had the following tumor sizes:

-   -   Animal 1: 1.68 cm³ tumor    -   Animal 2: 0.62 cm³ tumor    -   Animal 3: 1.22 cm³ tumor    -   Animal 4: 0.62 cm³ tumor    -   Animal 5: 0.56 cm³ tumor

The second pair of images shows the image at 1 hr post-peptideadministration. After only 1 hour the tumors in the mice pre-targetedwith the hBS14 were clearly visible. The majority of the signal waslocated in the bladder at this early time-point, as expected, and thekidneys also were evident in the images. External radioactivity wasfound on the foot of Animal 4 (circled) and was removed by washing thefoot.

The third pair of images show imaging data at 3 hrs post-peptideadministration. At the 3 hr time-point, Animal 3 was removed. This mousehad very high tumor uptake and adjusting the image for this mousedecreased the sensitivity in the remaining four mice. The outline of thetumors was visible in the mice that received only peptide, but this wasdue to the blood pool and not direct targeting as can be seen withAnimals 4 & 5. The mice were still under the effects of the anesthesiafrom the first time-point and were unable to void their bladders,resulting in the high signal observed in the bladded.

The final pair of images shows the image at 24 hrs post-peptideadministration. Little signal remained in the mice at this time-point,which therefore were imaged for 20 minutes rather than the 10 minutesused at earlier time-points. The only signal detected was located in thetumors of the mice pre-targeted with hBS14 prior to the administrationof the ^(99m)Tc-IMP-245.

All three pre-targeted mice and both mice that received peptide alonewere necropsied after the 24-hr imaging and the results are shown inFIG. 21 (bottom panel). Tumor and kidney uptake was the highest in thepre-targeted mice (19.01±2.80% ID/g and 3.81±0.80% ID/g, respectively).There was very little peptide in the tumor of the control mice(0.30±0.08% ID/g), but the same amount in the kidney as the pre-targetedmice (3.71±0.43% ID/g).

1. A polyvalent protein complex (PPC) comprising a first and a secondpolypeptide chain, wherein said first polypeptide chain comprises apolypeptide sequence represented, by the formula a₁-1₁-a₂-1₂-a₃, whereina₁, a₂, and a₃ are immunoglobulin variable domains and 1₁ and 1₂ arepeptide linkers, and a₁ is N-terminal of a₂, which in turn is N-terminalof a₃, wherein said second polypeptide chain comprises a polypeptidesequence represented by the formula b₁-1₃-b₂-1₄-b₃, wherein b₁, b₂, andb₃ are immunoglobulin variable domains and 1₃ and 1₄ are peptidelinkers, and b₃ is N-terminal of b₂, which in turn is N-terminal of b₁,wherein said first and second polypeptide chain together form a complexcomprising at least three antigen binding sites, wherein each of saidantigen binding sites comprises a variable domain from said firstpolypeptide chain and a variable domain from said second polypeptidechain, and wherein each binding site comprises an immunoglobulin heavychain variable domain and an immunoglobulin light chain variable domain.2. The complex according to claim 1 wherein each polypeptide chainfurther comprises 1-3 additional immunoglobulin variable domains,wherein each domain is linked via a peptide linker, wherein said firstand second polypeptide chain together form a complex comprising 4-6antigen binding sites, and wherein each of said antigen binding sitescomprises a variable domain from said first polypeptide chain and avariable domain from said second polypeptide chain.
 3. The complexaccording to claim 1, wherein at least one polypeptide chain furthercomprises an amino acid sequence selected from the group consisting of atoxin, a cytokine, a lymphokine, a enzyme, a growth factor, and anaffinity purification tag.
 4. The complex according to claim 1, whereinat least two of said antigen binding sites have the same bindingspecificity.
 5. The complex according to claim 1, wherein each of saidantigen binding sites has a different binding specificity.
 6. Thecomplex according to claim 4, wherein said antigen binding sites havethe same binding specificity.
 7. The complex according to claim 2wherein said antigen binding sites have at least two different bindingspecificities.
 8. The complex according to claim 7 wherein at least 3 ofsaid antigen binding sites have different binding specificities.
 9. Thepolyvalent protein complex of claim 7 wherein at least 4 of said antigenbinding sites have different binding specificities.
 10. The complexaccording to claim 7 comprising at least 5 antigen binding sites whereinat least 5 of said binding sites have different binding specificities.11. The complex according to claim 7 comprising 6 antigen binding siteseach having a different binding specificity.
 12. The complex accordingto claim 7 comprising at least 5 antigen binding sites wherein at least5 of said binding sites have different binding specificities.
 13. Thecomplex according to claim 1, wherein two of said antigen binding sitesare specific for epitopes of tumor associated antigens, and wherein saidthird antigen binding sites is reactive with a targetable construct. 14.The polyvalent protein complex of claim 13, wherein two antigen bindingsites are specific for epitopes of tumor associated antigens, andwherein the third antigen binding sites is reactive with a targetableconstruct, and wherein the epitope on the targetable construct is ahapten.
 15. A complex comprising at least one complex according to claim1 bound to a targetable construct, wherein said complex is bound to afirst hapten on said construct and wherein said construct furthercomprises a second hapten capable of binding simultaneously to a secondpolyvalent protein complex.
 16. The polyvalent protein complex of claim14, wherein the tumor associated antigen, or antigens are selected fromthe group consisting of antigens associated with carcinomas, melanomas,sarcomas, gliomas, leukemias and lymphomas.
 17. The polyvalent proteincomplex of claim 14, wherein the tumor associated antigen is selectedfrom the group consisting of α-fetoprotein, A3, CA125, carcinoembryonicantigen (CEA), CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45, CD74,CD80, colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, folatereceptor, HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia, IL-2,IL-6, insulin-like growth factor, KS-1, Le(y), MAGE, MUC1, MUC2, MUC3,MUC4, NCA66, necrosis antigens, PAM-4, placental growth factor,prostatic acid phosphatase PSA, PSMA, S100, T101, TAC, TAG-72, tenascinand VEGF.
 18. The polyvalent protein complex of claim 16, comprising atleast two tumor antigen binding sites, wherein both tumor antigenbinding sites are specific for CEA and wherein the third binding site isspecific for the hapten, histamine-succinyl-glycine (HSG).
 19. Thepolyvalent protein complex of claim 16, wherein the polyvalent proteinis BS14HP, or hBS14.
 20. A complex comprising a polyvalent proteincomplex according to claim 19, bound to IMP 241, or IMP 245
 21. Apretargeting method of treating or diagnosing or treating and diagnosinga neoplastic condition comprising (a) administering to said subject thepolyvalent protein complex of claim 1, wherein two antigen binding sitesare directed to a tumor associated antigen, and one antigen bindingsites is directed to a targetable construct comprising a bivalenthapten; (b) optionally, administering to said subject a clearingcomposition, and allowing said composition to clear the polyvalentcomplex from circulation; and (c) administering to said subject saidtargetable construct comprising a bivalent hapten, wherein saidtargetable construct further comprises one or more chelated orchemically bound therapeutic or diagnostic agents.
 22. The method ofclaim 21, wherein the diagnostic agent is a radionuclide selected fromthe group consisting of ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y,⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and ⁹⁰Y or a combination thereof.
 23. Themethod of claim 22, wherein said radioactive labels are imaged usingcomputed tomography (CT), single photon emission computed tomography(SPECT), or positron emission tomography (PET).
 24. The method of claim22, wherein the application is for intraoperative diagnosis to identifyoccult neoplastic tumors.
 25. The method of claim 21, wherein saidtargetable construct comprises one or more image enhancing agents foruse in magnetic resonance imaging (MRI).
 26. The method of claim 25,wherein said image enhancing agent is a metal selected from the groupconsisting of chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (III), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 27. The method ofclaim 21, wherein said targetable construct comprises one or more imageenhancing agents for use in ultrasound imaging.
 28. The method of claim21, wherein said targetable construct is a liposome with a bivalentHSG-peptide covalently attached to the outside surface of the liposomelipid membrane.
 29. The method of claim 28, wherein said liposome is gasfilled.
 30. The method of claim 21, wherein said targetable constructcomprises one or more radioactive isotopes useful for killing neoplasticcells.
 31. The method of claim 30, wherein said radioactive isotope isselected from the group consisting of ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁹⁰Y, ¹¹¹Ag, ₁₁₁In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac or acombination thereof.
 32. The method of claim 30, wherein the pretargetedtherapy is administered prior to, with or after one or more therapeuticagents.
 33. The method of claim 32, wherein said therapeutic agent is acytokine or a chemotherapeutic agent, or a colony-stimulating growthfactor.
 34. The method of claim 33, wherein said therapeutic agent is achemotherapeutic agent selected from the group consisting of taxanes,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas, triazenes; folic acid analogs, pyrimidine analogs, purineanalogs, vinca alkaloids, antibiotics, enzymes, platinum coordinationcomplexes, substituted urea, methyl hydrazine derivatives,adrenocortical suppressants, and antagonists.
 35. The method of claim33, wherein said therapeutic agent is a chemotherapeutic agent selectedfrom the group consisting of steroids, progestins, estrogens,antiestrogens, and androgens.
 36. The method of claim 33, wherein saidtherapeutic agent is a chemotherapeutic agent selected from the groupconsisting of azaribine, bleomycin, bryostatin-1, busulfan, carmustine,chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, dexamethasone,diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide,fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesteronecaproate, hydroxyurea, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,semustine streptozocin, tamoxifen, taxanes, taxol, testosteronepropionate, thalidomide, thioguanine, thiotepa, uracil mustard,vinblastine, and vincristine.
 37. The method of claim 33, wherein saidtherapeutic agent is a cytokine selected from the group consisting ofinterleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, interferon-alpha,interferon-beta, and interferon-gamma.
 38. The method of claim 33,wherein said therapeutic agent is a colony-stimulating growth factorselected from the group consisting of granulocyte-colony stimulatingfactor (G-CSF), granulocyte macrophage-colony stimulating factor(GM-CSF), erthropoietin and thrombopoietin.
 39. A method of treating aneoplastic disorder in a subject, comprising administering to saidsubject a “naked” polyvalent protein complex according to claim 1,wherein at least one of said antigen binding sites binds to an antigenselected from the group consisting of alpha fetoprotein, A3, CA125,carcinoembryonic antigen (CEA), CD19, CD20, CD21, CD22, CD23, CD30,CD33, CD45, CD74, CD80, colon-specific antigen-p (CSAp), EGFR, EGP-1,EGP-2, folate receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin,Ia, IL-2, IL-6, insulin-like growth factor, KS-1, Le(y), MAGE, MUC1,MUC2, MUC3, MUC4, NCA66, necrosis antigens, PAM-4, placental growthfactor, prostatic acid phosphatase PSA, PSMA, S100, T101, TAC, TAG-72,tenascin and VEGF.
 40. The method of claim 39, wherein the neoplasticdisorder is selected from the group consisting of carcinomas, sarcomas,gliomas, lymphomas, leukemias, and melanomas.
 41. A method for treatinga B-cell malignancy, or B-cell immune or autoimmune disorder in asubject, comprising administering to said subject one or more dosages ofa therapeutic composition comprising a polyvalent protein complex ofclaim 1 and a pharmaceutically acceptable carrier.
 42. A method fortreating a B-cell malignancy, or B-cell immune or autoimmune disorder ina subject, comprising administering to said subject one or more dosagesof a therapeutic composition comprising a polyvalent protein complex ofclaim 2 and a pharmaceutically acceptable carrier, wherein each antigenbinding site binds a distinct epitope of CD19, CD20 or CD22.
 43. Themethod of claim 42, wherein said polyvalent protein complex isparenterally administered in a dosage of 20 to 1500 milligrams proteinper dose.
 44. The method of claim 42, wherein said polyvalent proteincomplex is parenterally administered in a dosage of 20 to 500 milligramsprotein per dose.
 45. The method of claim 42, wherein said polyvalentprotein complex is parenterally administered in a dosage of 20 to 100milligrams protein per dose.
 46. The method of claim 42, wherein saidsubject receives the polyvalent protein complex as repeated parenteraldosages of 20 to 100 milligrams protein per dose.
 47. The method ofclaim 42, wherein said subject receives the polyvalent protein complexas repeated parenteral dosages of 20 to 1500 milligrams protein perdose.
 48. The method of claim 42, wherein a sub-fraction of thepolyvalent protein complex is labeled with a radioactive isotope. 49.The method of claim 48, wherein said radioactive isotope is selectedfrom the group consisting of ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y,¹¹¹Ag, ₁₁₁In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac or acombination thereof.
 50. A method for detecting or diagnosing a B-cellmalignancy, or B-cell immune or autoimmune disorder in a subject,comprising administering to said subject a diagnostic compositioncomprising a polyvalent protein complex of claim 2 and apharmaceutically acceptable carrier, wherein each antigen binding sitebinds a distinct epitope of CD19, CD20 or CD22, and wherein said complexis radiolabeled with a radionuclide selected from the group consistingof ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, 188Re,and ⁹⁰Y or a combination thereof.
 51. The method of claim 50, whereinsaid radioactive labels are imaged using computed tomography (CT),single photon emission computed tomography (SPECT), or positron emissiontomography (PET).
 52. The method of claim 50, wherein the application isfor intraoperative diagnosis to identify occult neoplastic tumors.
 53. Amethod for detecting or diagnosing a B-cell malignancy, or B-cell immuneor autoimmune disorder in a subject, comprising administering to saidsubject a diagnostic composition comprising a polyvalent protein complexof claim 2 and a pharmaceutically acceptable carrier, wherein eachantigen binding site binds a distinct epitope of CD19, CD20 or CD22, andwherein said complex is labeled with one or more image enhancing agentsfor use in magnetic resonance imaging (MRI).
 54. The method of claim 53,wherein said image enhancing agent is a paramagnetic ion selected fromthe group consisting of chromium (III), manganese (II), iron (II), iron(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium(III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 55. A method ofdiagnosing a non-neoplastic disease or disorder, comprisingadministering to a subject suffering from said disease or disorder acomplex according to claim 1, wherein a detectable label is attached tosaid complex, and wherein one or more of said antigen binding sites isspecific for a marker substance of the disease or disorder.
 56. Themethod of claim 55, wherein said disease or disorder is caused by afungus.
 57. The method of claim 56, wherein said fungus is selected fromthe group consisting of Microsporum, Trichophyton, Epidermophyton,Sporothrix schenckii, Cryptococcus neoformans, Coccidioides immitis,Histoplasma capsulatum, Blastomyces dermatitidis, and Candida albicans.58. The method of claim 55 wherein said disease or disorder is caused bya virus.
 59. The method of claim 58, wherein said virus is selected fromthe group consisting of human immunodeficiency virus (HIV), herpesvirus, cytomegalovirus, rabies virus, influenza virus, hepatitis Bvirus, Sendai virus, feline leukemia virus, Reo virus, polio virus,human serum parvo-like virus, simian virus 40, respiratory syncytialvirus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus,rubella virus, measles virus, adenovirus, human T-cell leukemia viruses,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus,wart virus and blue tongue virus.
 60. The method of claim 55 whereinsaid disease or disorder is caused by a bacterium.
 61. The method ofclaim 60, wherein said bacterium is selected from the group consistingof Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, and Mycobacterium tuberculosis62. The method of claim 55 wherein said disease or disorder is caused bya Mycoplasma.
 63. The method of claim 55 wherein said disease ordisorder is caused by a parasite.
 64. The method of claim 55 whereinsaid disease or disorder is malaria.
 65. The method of claim 55, whereinsaid disease or disorder is an autoimmune disease.
 66. The method ofclaim 65, wherein said autoimmune disease is selected from the groupconsisting of acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcalnephritis,erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosingalveolitis.
 67. The method of claim 55, wherein said the disease ordisorder is myocardial infarction, ischemic heart disease, oratherosclerotic plaques.
 68. The method of claim 55, wherein saiddisease or disorder is graft rejection.
 69. The method of claim 55,wherein said disease or disorder is Alzheimer's disease.
 70. The methodof claim 55, wherein said disease or disorder is caused by atopictissue.
 71. The method of claim 55, wherein said disease or disorder isinflammation caused by accretion of activated granulocytes, monocytes,lymphoid cells or macrophages at the site of inflammation, and whereinthe inflammation is caused by an infectious agent.
 72. The method ofclaim 55, wherein said detectable label is a radionuclide selected fromthe group consisting of ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd,¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and ⁹⁰Y or a combination thereof.
 73. The method ofclaim 72, wherein said radioactive labels are imaged using computedtomography (CT), single photon emission computed tomography (SPECT), orpositron emission tomography (PET).
 74. The method of claim 73, whereinthe application is for intraoperative diagnosis of said disease ordisorder.
 75. The method of claim 55, wherein at least one of saidantigen binding sites is specific for a targetable construct, andwherein said construct comprises one or more image enhancing agents foruse in magnetic resonance imaging (MRI).
 76. The method of claim 75,wherein said image enhancing agent is a paramagnetic ion selected fromthe group consisting of chromium (III), manganese (II), iron (III), iron(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium(III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 77. The method ofclaim 55, wherein at least one of said antigen binding sites is specificfor a targetable construct, and wherein said targetable constructcomprises one or more image enhancing agents for use in ultrasoundimaging.
 78. The method of claim 55, wherein at least one of saidantigen binding sites is specific for a targetable construct and whereinsaid targetable construct comprises a liposome with a bivalentHSG-peptide covalently attached to the outside surface of the liposomelipid membrane.
 79. The method of claim 74, wherein said liposome is gasfilled.
 80. A pretargeting method of treating or diagnosing anon-neoplastic disease or disorder in a subject comprising (a)administering to said subject the polyvalent protein complex of claim 1,wherein two antigen binding sites are directed to a marker substance, ormarker substances specific for the disorder, and one antigen bindingsites is directed to a targetable construct comprising a bivalenthapten; (b) optionally administering to said subject a clearingcomposition, and allowing said composition to clear the polyvalentcomplex from circulation; and (c) administering to said subject saidtargetable construct comprising a bivalent hapten, wherein thetargetable construct further comprises one or more chelated orchemically bound therapeutic or diagnostic agents.
 81. The method ofclaim 80, wherein said disease or disorder is caused by a fungus. 82.The method of claim 81, wherein the species of fungus is selected fromthe group consisting of Microsporum, Trichophyton, Epidermophyton,Sporothrix schenckii, Cryptococcus neoformans, Coccidioides immitis,Histoplasma capsulatum, Blastomyces dermatitidis, or Candida albicans.83. The method of claim 80 wherein said disease or disorder is caused bya virus.
 84. The method of claim 83, wherein the species of virus isselected from the group consisting of human immunodeficiency virus(HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningifis virus, wart virus and blue tongue virus.
 85. The methodof claim 80 wherein said disease or disorder is caused by a bacterium.86. The method of claim 85, wherein the bacterium is selected from thegroup consisting of Anthrax bacillus, Streptococcus agalactiae,Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli,Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilisinfluenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonasaeruginosa, Mycobacterium leprae, Brucella abortus, and Mycobacteriumtuberculosis.
 87. The method of claim 80 wherein said disease ordisorder is caused by a Mycoplasma.
 88. The method of claim 80 whereinsaid disease or disorder is caused by a parasite.
 89. The method ofclaim 80 wherein the disease or disorder is malaria.
 90. The method ofclaim 80, wherein said disease or disorder is an autoimmune disease. 91.The method of claim 90, wherein the autoimmune disease is selected fromthe group consisting of acute idiopathic thrombocytopenic purpura,chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura,post-streptococcalnephritis, erythema nodosurn, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pamphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis.
 92. The method of claim 80, wherein thedisease or disorder is selected from the group consisting of myocardialinfarction, ischemic heart disease, and atherosclerotic plaques.
 93. Themethod of claim 80, wherein the disease or disorder is graft rejection.94. The method of claim 80, wherein the disease or disorder isAlzheimer's disease.
 95. The method of claim 80, wherein the disease ordisorder is caused by atopic tissue.
 96. The method of claim 80, whereinthe disease or disorder is inflammation caused by accretion of activatedgranulocytes, monocytes, lymphoid cells or macrophages at the site ofinflammation, and wherein the inflammation is caused by an infectiousagent.
 97. The method of claim 80, wherein said targetable construct islabeled with a radionuclide selected from the group consisting of ¹⁸F,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷LU, ³²P, ¹⁸⁸Re, and ⁹⁰Y ora combination thereof.
 98. The method of claim 97, wherein saidradioactive labels are imaged using computed tomography (CT), singlephoton emission computed tomography (SPECT), or positron emissiontomography (PET).
 99. The method of claim 97, wherein the application isfor intraoperative diagnosis of the disorder.
 100. The method of claim80, wherein said targetable construct comprises one or more imageenhancing agents for use in magnetic resonance imaging (MRI).
 101. Themethod of claim 100, wherein image enhancing agent is a paramagnetic ionselected from the group consisting of chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III). 102.The method of claim 80, wherein said targetable construct comprises oneor more image enhancing agents for use in ultrasound imaging.
 103. Themethod of claim 102, wherein said targetable construct is a liposomewith a bivalent HSG-peptide covalently attached to the outside surfaceof the liposome lipid membrane.
 104. The method of claim 103, whereinsaid liposome is gas filled.
 105. A method of antibody dependent enzymeprodrug therapy (ADEPT) comprising; (a) administering to a patient witha neoplastic disorder the polyvalent protein complex of claim 3, whereinsaid complex comprises a covalently attached enzyme capable ofactivating a prodrug, (b) optionally administering to said subject aclearing composition, and allowing said composition to clear thepolyvalent complex from circulation, and (c) administering said prodrugto the patient.
 106. An assay method comprising detecting a targetmolecule using one or more polyvalent protein complexes of claim
 1. 107.An immunostaining method comprising staining a cell using one or morepolyvalent protein complexes of claim
 1. 108. An isolated nucleic acidmolecule encoding a first or second polypeptide according to claim 1.109. A nucleic acid expression cassette comprising the isolated nucleicacid of claim
 108. 110. An episome comprising: (a) a first promoteroperationally connected to a first nucleic acid encoding a firstpolypeptide comprising a polypeptide chain represented by the formulaa₁-1₁-a₂-1₂-a₃, wherein a₁, a₂, and a₃ are immunoglobulin variabledomains and 1₁ and 1₂ are peptide linkers, (b) a second promoteroperationally connected to a second nucleic acid encoding a polypeptidecomprising a second polypeptide chain represented by the formulab₁-1₃-b₂-1₄-b₃, wherein b₁, b₂, and b₃ are immunoglobulin variabledomains and 1₃ and 1₄ are peptide linkers, wherein said first and secondpolypeptide chain together form a complex comprising at least threeantigen binding sites, wherein each of said antigen binding sitescomprises a variable domain from said first polypeptide chain and avariable domain from said second polypeptide chain, wherein said firstnucleic acid and said second nucleic acid are coexpressed when theepisome is transformed into a host cell.
 111. The episome of claim 110which is a plasmid or a cosmid.
 112. A host cell comprising an episomeaccording to claim
 110. 113. The host cell of claim 112, wherein saidhost cell is selected from the group consisting of E. coli, yeast, aplant cell and a mammalian cell.
 114. A method of preparing a polyvalentprotein complex, comprising culturing a host cell according to claim112.
 115. The host cell of claim 112, wherein said cell is a murinemyeloma cell line.
 116. The episome of claim 111, wherein the plasmid ispdHL2.